Microscopy, Immunohistochemistry, and Antigen Retrieval Methods

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Microscopy, Immunohistochemistry, and Antigen Retrieval Methods
For Light and Electron Microscopy

M. A. Hayat
Kean University Union, New Jersey

KLUWER ACADEMIC PUBLISHERS
NEW YORK, BOSTON, DORDRECHT, LONDON, MOSCOW

eBook ISBN: Print ISBN:

0-306-47599-5 0-306-46770-4

©2002 Kluwer Academic Publishers New York, Boston, Dordrecht, London, Moscow Print ©2002 Kluwer Academic/Plenum Publishers New York All rights reserved No part of this eBook may be reproduced or transmitted in any form or by any means, electronic, mechanical, recording, or otherwise, without written consent from the Publisher Created in the United States of America Visit Kluwer Online at: and Kluwer's eBookstore at: http://kluweronline.com http://ebooks.kluweronline.com

To my friends for their generosity

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Preface

There are several important reasons for publishing this book. One reason is to present chemical and physical principles governing the processing of tissues using microwave heating as an adjunct to fixation, embedding in a resin, and staining. A second reason is to point interested readers to a number of recent developments in the retrieval and localization of antigens in normal and pathological tissues. The greatest concentration of work in this field has focused on the detectability of disease-related proteins. Therefore, as examples, the detectability and the role in disease of estrogens, p53, p185, Ki-67, and PCNA are discussed in detail. A third reason is to review favorable aspects of the histochemical approach, whereby it yields data not obtainable by any other means, including biochemical assays. Histochemistry, for instance, contributes to acquiring knowledge about the biological activity of normal and diseased cells, which is supported by illustrations. Immunohistochemistry defines the function of cell types in a tissue and organs by localizing and identifying their contents or products. This methodology is highly visual; illustrations, especially color images, often contribute as much to correct understanding and interpretation of the results as the text. Therefore, the results of many methods are illustrated. During the last decade there has been significant progress in understanding the mechanisms responsible for antigen masking during fixation and subsequent unmasking, primarily by heating or, in some cases, by enzymatic digestion. Comparative studies demonstrate, for example, that not only microwave heating but also other sources of heating are effective in antigen retrieval. Similar studies also indicate that although sodium citrate buffer is in common use as the antigen retrieval fluid, unmasking of certain antigens requires other fluids. These and other new developments are discussed in this volume. In preparing the reader to study the location of proteins and carbohydrates, it is necessary to explain the advantages and limitations of the study. A potential limitation of the immunohistochemical approach arises from the possibility of false-negative staining due to the failure of an antibody to yield positive results. It is equally important to be aware of the possibility of false-positive staining, which can arise if the method is not scrupulous regarding histochemical negative and positive controls. Suggestions are offered to at least minimize these histological artifacts. In this regard the importance of negative and positive controls cannot be overemphasized. Negative controls involve the omission of the primary
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antibody with an immunoglobulin that is directed against an unrelated antigen. This immunoglobulin must be of the same class, source, and species. Positive controls involve the use of a tissue section of known positivity. The absence of staining in a test tissue section does not necessarily indicate that the antigen is not present. It should be noted that some antigens are present not only in pathological tissues but also in healthy tissues. The arrival of methods and instruments to investigate disease processes at the molecular level induces pathologists to apply these new procedures to existing problems of disease pathogenesis and disease evolution and offers clues to therapeutic intervention. Such methods include histological microdissection (in conjunction with real-time quantitative reverse transcriptase–polymerase chain reaction), cDNA microarray, anticancer vaccines, and gene regulation (genes can be turned on and off). Some of these techniques are summarized in Chapter 1. The limited space available did not allow a detailed presentation of the expanding world of molecular pathology. Chapter 1 contains seemingly diverse topics, but all are related to immunopathology. It is my hope that pathologists will benefit from these step-by-step protocols, which are presented in a self-explanatory form so that the reader can practice them without outside help. Chapter 8 contains details of specific methods because the various parameters of processing of each type of antibody, antigen, and tissue may need to be varied to obtain optimal results. I have tried to synthesize a large number and variety of immunohistochemical techniques into a single and concise basic handbook. Some alternative methods are also included. I have explained not only how to use a technique but also why to use it—along with its advantages and limitations. An example is the microwave heating methodology. The methods presented were carefully selected and are reproducible but can be modified, depending on the objective of the study. Moreover, as the antigen retrieval methodology is relatively new, it requires fine-tuning. There is a degree of necessary repetition in some chapters, which allows them to stand alone. This approach helps the reader to carry out a procedure where it is described without searching for its details somewhere else in the book. Cross-reference of information among chapters, wherever possible, is given. Where possible, commercial sources of reagents, kits, and equipment are listed throughout the text instead of in a separate index. Extensive references are provided to facilitate the task for those readers who may wish to consult the literature for additional information on specific topics. All books can be improved, and this volume is no exception. I welcome constructive criticism from my colleagues and students. With this help I look forward to offering a greatly improved second edition. The writing of this book would not have been possible without the most generous help of a large number of distinguished scientists. I am very grateful for the thoughtful and invaluable suggestions and illustrations received from scientists throughout the world. It is appropriate to acknowledge significant contributions made to the understanding and practice of antigen retrieval methodology and applications of microwave heating by Hector Battifora, Mathilde E. Boon, Giorgio Cattoretti, Richard J. Cote, Ann M. Dvorak, Jules M. Elias, Johannes Gerdes, David Hopwood, Allen M. Gown, Richard Horobin, L. P. Kok, Anthony S.-Y. Leong, Gary R. Login, Enrico Marani, Shan-Rong Shi, Albert J. H. Suurmeijer, and Clive R. Taylor. It is not possible to mention all the scientists who have played a role in the development of this technology.

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The help and encouragement received from Dean Betty Barber throughout the writing of this book are greatly appreciated and will be remembered. I thank Patricia Lemus and Elizabeth McGovern for their expert secretarial assistance in the preparation of the manuscript, and I appreciate the help and cooperation extended to me by Roberta Klarreich, the production editor, throughout the production of this volume. M. A. Hayat October 2001

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Contents

Chapter 1. Introduction
Cytogenetic Evaluation of Tumors Genetic Instability of Tumors Tumor Heterogeneity
Histological Microdissection

1
11 12 14 14 15 17 18 20 23 24 25 28 29

Antitumor Vaccines Molecular Genetics cDNA Microarray Technology Angiogenesis
Vascular Endothelial Growth Factor Immunohistochemical Localization of Vascular Endothelial Growth Factor Telepathology (Telemedicine)

Future of Immunohistopathology Preparation of Buffers

Chapter 2. Antigens and Antibodies
Antigens
Epitopes

31
31 32 33 34 35 35 37 38 39 41 44 46 47
xi

Antibodies
Polyclonal Antibodies Production of Polyclonal Antiserum Affinity Chromatography Monoclonal Antibodies Specificity of Monoclonal Antibodies MIB-1 Monoclonal Antibody Production of Monoclonal Antibodies Bivalent and Bispecific Monoclonal Antibodies in Cancer Therapy

Recombinant Antibodies Anticancer Monoclonal Antibodies

xii

Contents

Antibody Cross-Reactivity Polyreactive Antibodies Commercial Sources of Antibodies

48 49 50

Chapter 3. Fixation and Embedding
Formaldehyde
Nature of Formaldehyde Solution Mechanism of Fixation with Formaldehyde Comparison of Formaldehyde with Glutaraldehyde Fixation with Formaldehyde Effect of Prolonged Fixation with Formaldehyde Formalin Substitute Fixatives Fixation Conditions

53
53 54 54 56 57 58 59 60 61 62 64 64 65 65 67 67 67 68 69

Effect of Heating on Fixation with Glutaraldehyde Microwave Heat–Assisted Fixation with Osmium Tetroxide Role of Microwave Heating in Enzyme Cytochemistry
Fixation for Enzyme Cytochemistry Using Microwave at Relatively Low Temperature

Cryopreservation in the Presence of Microwave Heating Paraffin Embedding
Paraffin Embedding in Microwave Oven Paraffin Embedding in Vacuum-Microwave Oven Microtomy of Paraffin-Embedded Tissues Silanting of Glass Slides Vacuum-Assisted Microwave Heating

Chapter 4. Factors Affecting Antigen Retrieval
Fixation Denaturation Heating pH Molarity Antigen Retrieval Fluids
Glycerin as Antigen Retrieval Fluid Procedure pH of Antigen Retrieval Fluids Ionic Strength of Antigen Retrieval Fluids

71
71 72 73 74 74 75 77 78 78 79 79 80 82 83 84 89

Antibody Penetration Antibody Dilution
Diluent Buffer for Primary Antibodies

Storage of Paraffin-Embedded Tissues Storage of Tissue Slides Signal Amplification

Contents Tyramine Amplification Method Preparation of Biotinylated Tyramine Rolling Circle Amplification

xiii

90 92 92

Chapter 5. Problems in Antigen Retrieval
Lack of Immunostaining Background Staining Problem of Endogenous Biotin
Procedure

95
95 96 98 100 101 101 102 102 103 104 105 105 107 109 109 109 110 111 113 114

Mirror Image Complementary Antibodies
Procedure

Fixation of Frozen Tissues Hot Spots (Areas) in Microwave Oven Problem of Antigen Retrieval Standardization Test Battery Intraobserver and Interobserver Variation in Diagnosis Quantitation of Immunostaining Autostainers
Capillary Gap Stainers Centrifugal Stainers Flat-Method Stainers

Volume-Corrected Mitotic Index The Gleason Grading System Universal Antigen Retrieval Method? Calibration of Microwave Oven

Chapter 6. Antigen Retrieval
Possible Mechanisms of Antigen Retrieval
Nonthermal Effects of Microwave Heating

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117 119 120 123 124 124 125 130 131 132 132 136 138 139 139 141

Effect of Endogenous Calcium on Antigen Masking Use of Ethylenediaminetetraacetic Acid (EDTA) for Antigen Retrieval Antigen Retrieval with Heat Treatment Advantages of Heating Heating Methods
Mechanism of Epitope Retrieval by Microwave Heating Duration of Microwave Heating Antigen Retrieval in a High-Pressure Microwave Oven Antigen Retrieval at Low Temperature Use of Heat for Staining Rapid Immunostaining of Frozen Sections Enhanced Polymer One-Step Staining Procedure Modified En Vision Procedure Hazards and Precautions in the Use of Microwave Ovens

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Contents

Limitations of Microwave Heating

Wet Autoclave Method
Procedure 1 Procedure 2

Ultrasound Treatment
Procedure

Nonheating Methods
Detergents Procedures Proteolytic Enzyme Digestion Procedure Enzyme Digestion and Relatively Low Temperature (80°C)–Assisted Antigen Retrieval

142 145 145 146 146 148 148 148 149 151 152 152 153

Comparison of Antigen Retrieval Methods: A Summary

Chapter 7. Antigen Retrieval on Resin Sections ....................
Role of Fixative and Embedding Resin in Antigen Retrieval Immunostaining of Thin Resin Sections Antigen Retrieval on Sections of Modified Epoxy Resin Effect of Heating Antigen Retrieval on Thin Resin Sections Using Autoclaving Rapid Staining of Thin Resin Sections in Microwave Oven Microwave Heat–Assisted Rapid Processing of Tissues for Electron Microscopy Microwave Heat–Assisted Immunolabeling of Resin-Embedded Sections Microwave Heat–Assisted Immunogold Methods
Immunogold-Silver Staining Droplet Procedure

155
156 158 160 161 161 163 163 163 167 167 167

Chapter 8. General Methods of Antigen Retrieval
General Procedure for Antigen Retrieval Using Microwave Heating Antigen Retrieval in Archival Tissues
Method for Microwave Heating of Archival Tissue Blocks

169
169 173 174 175 175 175 176 177 178 178 180 180

Antigen Retrieval Using a Conventional Oven Hot Plate–Assisted Antigen Retrieval
Procedure

Hot Plate–Assisted Grading of Vulvar Intraepithelial Neoplasia Water Bath Heat–Assisted Antigen Retrieval
Procedure for Electron Microscopy Procedure for Light Microscopy Procedure for Free-Floating Sections

Microwave Heat–Assisted Evaluation of Global DNA Hypomethylation

Contents Procedure

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181 181 181 182 182 183 183 184 185 186 186 186 187 187 188 189 189 190 191 191 192 192 192 194 194 196 196 197 198 198 199 199 200 200 201

Microwave Heat–Assisted Enhanced Peroxidase One-Step Method
Procedure

Microwave Heat–Assisted Immunostaining of Cell Smears Double Immunostaining Using Microwave Heating Microwave Heat–Enhanced Double Immunostaining of Nuclear and Cytoplasmic Antigens
Procedure

Microwave Heat–Assisted Immunohistochemical Localization of Cyclin D1 Microwave Heat–Assisted Immunofluorescence Staining of Tissue Sections
Procedure

Microwave Heat–Assisted Double Immunofluorescence Labeling
Procedure

Microwave Heat–Assisted Double Indirect Immunofluorescence Staining
Procedure Control Procedures Immunoenzymatic Detection

Combined Microwave Heating and Ultrasound Antigen Retrieval Method Combined Enzyme Digestion and Microwave Heating Antigen Retrieval Method Pressure Cooker–EDTA–Assisted Antigen Retrieval 2-Mercaptoethanol–Sodium Iodoacetate–Assisted Antigen Retrieval Antigen Retrieval with Steam–EDTA–Protease Method
Procedure

Picric Acid–Steam Autoclaving–Formic Acid–Guanidine Thiocyanate–Assisted Retrieval of Prion Protein
Procedure

Simultaneous Detection of Multiple Antigens
Procedure

Use of Multiple Antibodies for Labeling Antigens
Procedure

Antigen Retrieval in Neuronal Tissue Slices before Vibratome Sectioning Microwave Heat–Assisted Antigen Retrieval in Freshly Frozen Brain Tissue
Procedure

Microwave Heat–Assisted Rapid Immunostaining of Frozen Sections
Procedure

Microwave Heat–Assisted Immunocytochemistry of Thin Cryosections
Procedure

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Contents

Pressure Cooker–Assisted Detection of Apoptotic Cells Immunohistochemical Localization of Prostate-Specific Antigen Immunohistochemistry
Procedure

201 202 203 203

Chapter 9.

Other Applications of Microwave Heating
Carbohydrate Antigens
Ovarian Carcinoma Microwave Heat–Assisted Carbohydrate Antigen Retrieval Enzyme Digestion–Assisted Carbohydrate Antigen Retrieval

205
205 206 208 208 209 211 212 213 215 215 217 218 219 221 221 221 222 222 223 224 224 225 225 227 228 228 229 230 230 230

Nucleolar Organizer–Associated Region Proteins
Procedure Nucleolar Size

In Situ Hybridization
Radioactive Probes Nonradioactive Probes Enhancement of in Situ Hybridization Signal with Microwave Heating

Procedure for in Situ Hybridization of DNA In Situ Hybridization of RNA in Skeletal Tissues Microwave Heating for in Situ Hybridization of mRNA in Plant Tissues Microwave Treatment Staining Microwave Heat–Assisted Fluorescence in Situ Hybridization Procedure for Gastrointestinal Neoplasia Nuclear Fluorescence in Situ Hybridization Signal Using Microwave Heating

Microwave Heat–Assisted Polymerase Chain Reaction
Procedure

Detection of Antigens by Flow Cytometry Microwave Heat–Assisted Flow Cytometry
Procedure 1 Procedure 2

Microwave Heat–Assisted Microwave Heat–Assisted Microwave Heat–Assisted Microwave Heat–Assisted
Procedure

Enzyme-Linked Immunosorbent Assay Scanning Electron Microscopy Confocal Scanning Microscopy Correlative Microscopy

Chapter 10. Cell Proliferating Antigens
Ki-67 Antigen
Immunohistochemistry Limitations of Immunohistochemistry Antibodies

233
233 235 237 237

Contents Recent Applications of MIB-1 Antibody Ki-67 Antigen Retrieval Using Microwave Heating Ki-67 Antigen Retrieval Using Autoclave Treatment

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Proliferating Cell Nuclear Antigen
Immunohistochemistry Limitations of PCNA Immunohistochemistry Immunostaining of PCNA on Cryostat Sections

p53 Antigen
Wild-Type p53 Protein Mutant p53 Protein p73 Antibodies Examples of Antibody Dilutions Immunohistochemistry

Use of Multiple Antibodies for Labeling p53 Antigen
Wild-Type p53 Antigen Retrieval Using Microwave Heating p53 Antigen Retrieval Using Microwave Heating Frozen Section Immunohistochemistry of p53

239 240 240 241 242 243 245 245 247 248 249 250 253 253 256 257 257 258

Chapter 11. Estrogens
Estrogen Receptors
Estrogen Receptor Alpha Estrogen Receptor Beta Estrogen Receptor Gamma Distribution of Estrogen Receptors

261
262 265 267 268 268 269 270 270 273 275 276 276 277 278

Role of Estrogen Receptors in Breast Cancer
Breast Cancer and Tamoxifen

Antibodies Immunohistochemistry Comparison of Immunohistochemistry with Biochemical Ligand-Binding Assays
Dextran-Coated Charcoal Assay

Semiquantitative Assessment of Estrogen Receptors Immunostaining of Estrogen Receptors in Prostate Tissue Immunostaining of Estrogen and Progesterone Receptors in Fine-Needle Aspirates of Breast

Chapter 12. HER-2 (c-erbB-2) Oncoprotein
HER-2/neu Oncogene HER-2 Oncoprotein HER-2 Overexpression Simultaneous Overexpression of HER-2 and p53 Distribution of HER-2 in Carcinomas

281
281 283 284 284 285

xviii Astrocytic Tumors Bladder Carcinoma Ewing’s Sarcoma Intrahepatic Cholangiocellular Carcinoma Laryngeal Squamous Cell Carcinoma Non-Small-Cell Lung Carcinoma Ovarian Carcinoma Prostate Carcinoma Squamous Cell Carcinoma of Cervix

Contents

Methods for Detecting HER-2 Status Quantitative Analysis of HER-2/neu Gene Expression Detection of HER-2 Oncoprotein Bispecific Antibodies
Bispecific Antibody MDX-H210

Vaccines
Genetic Immunization

Immunohistochemistry Herceptin (Trastuzumab) HercepTest
Controls and Scoring System

Immunostaining of HER-2 Protein Using HercepTest

285 285 285 286 286 286 287 288 288 289 290 291 293 294 295 295 296 297 299 300 302

References Index

305 351

Microscopy, Immunohistochemistry, and Antigen Retrieval Methods

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Chapter 1

Introduction

Immunohistochemistry and immunocytochemistry have played an important role in the fields of cell and tissue biology, embryology, and diagnostic pathology. These methodologies facilitate precise analysis of the chemistry of cells and tissues in relation to structural organization. The information derived from these techniques will continue to contribute to our understanding of dynamic molecular, cellular, and pathological processes. For example, immunohistochemistry has revolutionized the field of tumor diagnosis and has provided a powerful tool for pathologists to better characterize difficult or unusual neoplasms. In addition, this technology has provided information that has resulted in the reclassification of many neoplasms and, in some cases, the creation of new categories that were previously unrecognized. In this volume the emphasis is on the application of this methodology to routine diagnostic pathology. The immunohistochemical method localizes and identifies a specific antigen in a cell or a tissue specimen. In the most common approach, specimens are fixed in 10% neutral buffered formalin and embedded in paraffin. Sections ( thick) are deparaffinized with xylene and then rehydrated in a series of ethanol solutions with decreasing concentrations. After drying, sections are treated with 3% hydrogen peroxide to block endogenous peroxidase. If required, sections are subjected to antigen retrieval. Nonspecific immunostaining is blocked by treating the sections with 10% normal serum. The primary antibody applied is either monoclonal or polyclonal. A secondary antibody, linked with an imaging system (usually a peroxidase), is applied to recognize the primary antibody. Contrary to the popular use of the term antigen retrieval in the literature and by commercial companies, the correct term is epitope retrieval, since it is the epitope (antigenic determinant) that is recognized by the antibody (paratope) instead of antigen molecule as a whole. A monoclonal antibody reacts with a specific region of the antigen irrespective of the conformation of other regions of the same antigen. Different monoclonal antibodies generally react with different epitopes of the same antigen. Thus, the epitope is the part of the antigen to which the antibody is directed. An epitope indeed defines antigen specificity. Furthermore, the fact that monoclonal antibodies to different epitopes of the same antigen molecule behave differently in “antigen retrieval” indicates that what is being retrieved is an epitope. In other words, two different epitopes of the same antigen may require different treatments for their accessibility to the respective antibodies. This is the context in which this subject should be understood.
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The other point of view, which favors the term antigen retrieval instead of epitope retrieval, is as follows (S.-R. Shi, personal communication). It is likely that the mechanism of antigen retrieval is based on chemical modification of protein conformation. Therefore, retrieval of formalin-modified (or masked) antigenicity must be a restoration of the protein structure, as any antigen/antibody recognition is dependent on protein conformation. This is particularly true for discontinuous epitopes (most antigen determinants are discontinuous epitopes), which consist of amino acid sequences apart from each other on one polypeptide (or actually located on distinct polypeptides) but brought near each other in the tertiary or quaternary structure of the protein. In other words, restoration of the function of an epitope (antigenicity) is the retrieval of its protein conformation, i.e., retrieval of antigen. Because the concept of epitope is not an intrinsic feature of a protein existing independently of its paratope partner, the term epitope refers to only a functional unit, but not a stoic structure of the protein. Shi et al. (2000a) have further justified the use of the term antigen and rejected the relevancy of the term epitope in immunohistochemistry. In support of Dr. Shi’s opinion is the fact that in some cases the absolute specificity of even monoclonal antibodies can be questioned. The absorption control cannot always determine whether the protein bound in the tissue is the same protein used for absorption. The monoclonal antibody may instead recognize a similar epitope of an unrelated protein, especially following tissue fixation. Absorption controls therefore may not provide the specificity of the antibody for a protein under study in the tissue. In light of the above-mentioned difference of opinion, and in the absence of a definite understanding regarding unmasking of an epitope or whole antigen molecule as a result of unmasking treatments (heat or nonheat treatment), both terms, epitopes and antigens, are used in this volume. Immunohistochemistry has surpassed other techniques in its effectiveness in the in situ preservation and detection of antigens. Immunopathology has become a valuable or even an essential adjunct to diagnostic pathology. It is affirmed that diagnostic immunohistochemistry is indispensable in surgical pathology for diagnosis, therapy, and prognosis. The usefulness of this methodology depends and will continue to rely on three major factors: (1) availability of specific primary antibodies; (2) an efficient detection system; and (3) correct interpretation and significance of the findings. An increasing number of monoclonal antibodies is being produced, and many of them are commercially available; these sources are given in Chapter 2. The role of antibodies in diagnostic pathology is discussed in this chapter. Highly sensitive detection systems are available, and their signals are being continuously enhanced to achieve high signal-to-noise ratio. Methods of scoring the signals are also available. These improvements are discussed in Chapter 4. All immunohistological methods depend on the successful completion of a series of sequential steps, beginning with specimen collection; their morphological and antigenic preservation (chemical fixation or rapid freezing) or antigenic retrieval; incubation in an antibody or sequence of antibodies; staining; signal counting; and interpretation of results. Each of these steps must be performed as efficiently and correctly as possible. If even one of these steps is suboptimal, the remaining steps, even though perfectly carried out, can never compensate for the inefficient step. Any error in immunohistochemistry, especially when applied to clinical diagnosis, is unacceptable. This methodology is not only a science but also an art; each aspect depends on the other. Progress in molecular biology is intimately related to advancement in technology. One fundamental goal of cell research is to understand the functions of molecules that

Introduction

3

constitute cells and tissues. This understanding can be enhanced by examining the molecular details and subcellular location of cell components. The precise extracellular and intracellular localization of molecules under different physiological and pathological conditions yields clues to their possible functions. These aspects of antigen molecules and receptors, especially clinically important ones such as p53, Ki-67, PCNA, p185, and estrogens, are discussed in detail in Chapters 10, 11, and 12. The achievement of the above-mentioned goal received an impetus from the development of the heating methodology (especially microwave heating) for antigen retrieval. Microwave heating was introduced into biomedical research approximately two decades ago for the rapid processing of plant and animal tissues. The development of this technique was a significant step forward in the application of histochemistry, immunohistochemistry, and immunocytochemistry. In other words, this methodology has significantly contributed to the localization of macromolecules and molecules (including antigens) and thus to an understanding of their functions. This technique is also useful for enhancing the detection of RNA and DNA by in situ hybridization (see page 213). Another example of the application of microwave heating is in conjunction with flow cytometry (see page 225). The polymerase chain reaction (PCR) has also been used in conjunction with microwave heating for studying DNA (see page 224). Yet another application of microwave heating is with the enzyme-linked-immunosorbent assay method (ELISA) (see page 228). Tissue cryopreservation with diminished ice crystal growth has also been accomplished with this versatile technology (see page 65). Application of microwave heating to enzyme cytochemistry, autoradiography, and X-ray microanalysis has been attempted (Mizuhira and Hasegawa, 1996). Significant aspects of the basic biology of disease processes are now assuming clinical importance in diagnosis and prognosis. The pathologist can identify an ever-increasing range of antigens in tissue sections using the techniques mentioned above. Identification of tissue antigens using these methods is of fundamental importance for clarifying tumor proteins or carbohydrates, determining the diagnosis and prognosis of tumors, characterizing pervasive nepotistic alterations in tissues such as prostate, subclassifying neoplasms, evaluating the response of tumors and pervasive nepotistic changes to certain therapies (i.e., as a surrogate intermediate and end point), selecting patients who are candidates for specific therapies (e.g., immunotherapy), and identifying pathogenic organisms (Arnold et al., 1996). For these and other reasons, immunohistochemistry has become the most important tool in research and diagnostic pathology. It permits detection of defined antigens on cryostat, paraffin, and resin sections of normal and diseased tissues. Immunohistochemical and immunocytochemical localization of antigens is a powerful tool that provides insight into some of the salient features of cell and tissue complexity. Such studies, for example, demonstrate relationships between normal cell structure and function and pathological consequences. Presently, immunohistochemistry is firmly established as the most important method for detecting antigens with the light microscope. It can be effectively used to examine various antigens in the sections of formaldehyde-fixed and paraffin-embedded tissues (Hayat, 2000a). The availability of the equipment to carry out immunohistochemistry and the introduction of many new monoclonal antibodies make it possible to apply this technique to retrospective studies. The introduction of a large number of new monoclonal antibodies of improved sensitivity and specificity, which are available in ready-to-use kits, has made possible a wider use of immunohistochemistry for antigen analysis. In addition, the development of various

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Chapter 1

antigen retrieval methods during the past two decades has enabled many more antibodies to access antigens that were undetectable or minimally detectable in the past. Today almost any antigen that survives tissue processing has the potential to be localized immunohistochemically. As a result of these methods, additional antibodies have become paraffin- and resin-compatible, which permits heat-treated tissue sections to be used for detecting antigens with the light and electron microscopes. New antigen retrieval methods, especially microwave heating and other heating procedures and ultrasound treatment, can effectively retrieve antigens from tissues left in formaldehyde for prolonged periods. The introduction of the computer-assisted image analyzer and other automated equipment (e.g., the automatic stainer), and generation of antibodies to synthetic peptides, have ushered immunohistochemistry into a higher level of efficiency, accuracy, and quantitation. The demand for a more precise spatial localization of epitopes favors the use of antibody fragments (e.g., Fab), peptides, or ligands. These advances facilitate the use of antigen detection for correct diagnosis and prognosis. Furthermore, advances in detection accuracy provide guidelines to study and understand more complicated biological problems. To achieve these goals, standardization of antigen retrieval methods is necessary, at the least, to minimize inter- and intralaboratory (including interobserver) variability of immunostaining (see Chapter 5). However, even in the absence of such standardization, the method has become the most effective tool in light microscope immunohistochemistry and, to some extent, in electron microscope immunocytochemistry.

Introduction

5

Although antigen retrieval is carried out most commonly on paraffin sections, it can also be accomplished on semithin or thin resin sections for light and electron microscopy, respectively. Thin sections of routinely used resins such as epoxy, LR White, LR Gold, and Lowicryls can be used for detecting antigens with the light or electron microscope. These resins, in conjunction with microwave heating, can also be used for cell and ultrastructural studies with the light microscope, and scanning and transmission electron microscope (Fig. 1.1). This procedure can also be employed for studying bacteria with the scanning electron microscope (Fig. 1.2). The advantages of resin sections include better preservation of cellular details, assisting the achievement of higher resolution, and the ability to carry out correlative studies of the same tissue with the light microscope and scanning and transmission electron microscope (Fig. 1.3). In addition, resin sections (also cryosections) allow immunogold and silver-enhanced immunogold staining. Biochemical assays such as the dextran-coated charcoal (DCC) assay, certain signal amplification techniques, and other cytosol-based methods have been mostly replaced

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Chapter 1

by immunohistochemistry because the former methods are costly and often difficult to reproduce. For example, the DCC assay requires rather large tissue specimens and may be adversely influenced by tissue heterogeneity (e.g., tumors), presence of bound endogenous estrogen, and sampling error (Hendricks and Wilkinson, 1993). In cytosol-based biochemical assays, tissues are indiscriminately homogenized (e.g., tumor, stroma, inflammatory cells, and epithelial cells). Therefore, the results expressed in fentomoles per milligram of the total protein are variably diluted because of the presence of nontumorous cells. In contrast, immunohistochemistry can be carried out with smaller tissue specimens and is less affected by tissue heterogeneity or endogenous hormones. This technique allows direct histological visualization, which permits separation of tumors from stroma, inflammatory cells, and normal cells. However, the DCC assay can be employed for correlative studies; therefore, the selection of a particular method in a diagnostic laboratory should depend on a number of factors, including specificity, sensitivity, rapidity, use of potentially harmful reagents, availability of equipment, cost, and application to a wide range of antigens. However, the central problem in immunohistochemistry is to retain antigenicity without sacrificing the quality of cell morphological preservation. It has been established that the preservation of antigenicity is inversely related to the preservation of cell morphology (Hayat, 2000a); thus, tissue preparation methods optimal for the preservation of cell morphology introduce protein crosslinking and are therefore suboptimal for preserving antigenicity. Because preserving antigenicity in immunohistochemistry is more important than preserving morphology, 10% formaldehyde is generally used for fixation. Glutaraldehyde, on the other hand, yields excellent ultrastructural preservation but severely masks most antigens by introducing irreversible protein crosslinking. The mechanism(s) responsible

Introduction

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8

Chapter 1

for the antigen-masking effects of fixatives are discussed in Chapter 4. Tissue embedding in paraffin also adversely affects antigen detection. The alternative to chemical fixation and paraffin embedding is to use frozen sections of fresh-frozen tissues (snap freezing). These cryostat sections tend to provide improved antigen detection. Proteins are retained in these sections at least until the cryosections are placed into aqueous incubation and staining solutions. Most antibodies recognize antigens better in frozen sections than in sections of chemically fixed and paraffin-embedded tissues. Fixation of frozen sections has also been recommended by some workers to immobilize proteins during subsequent processing of cryostat sections and thus improve the antigen localization. However, an agreement on the beneficial effect of this practice is lacking. Moreover, cryostat sections are difficult to prepare, and the quality of morphological preservation is comparatively poor (Fig. 1.4). Fortunately, methods are available that satisfactorily preserve cell morphology as well as antigenicity. The best compromise is to use a mixture of 6% formaldehyde and glutaraldehyde of a low concentration (0.1–0.4%). This approach, which is used extensively for electron microscopy, should be used for light microscopy and is presented in this volume. The optimal immunohistochemical method should ideally detect all specific epitopes, but in practice this is not possible. The best that can be accomplished is to optimize the protocol to detect all the detectable epitopes. Presently, the majority of the antigen retrieval studies are carried out using microwave heating, although other heating methods such as autoclaving (page 145) or pressure cooking (pages 127–128) can be equally effective, depending on the types of tissue and antigen under study. Antigen retrieval can also be accomplished in a microwave oven under vacuum, as can rapid fixation, resin embedding, and staining. Ultrafast (milliseconds) or fast (seconds to minutes) fixation can be achieved with this method. Even rapid fixation with osmium tetroxide can be carried out in a microwave oven (see Chapter 3). Like any other technique, microwave heating has certain limitations. A well-known example is the uneven distribution of hot and cold spots in the oven, although this can be minimized by using a water load in the oven. Prior to placing the water load in the oven, cold and hot spots should be located in the oven cavity by using a high-brightness neon bulb array. Hot spots must be avoided because an excessive increase in temperature during irradiation is a major cause of poor fixation. The problem of hot spots apparently can be avoided by using heating methods other than microwave heating. One should also be aware that microwave heating may promote cross-reactivity by producing or unmasking antigens closely related to those under study. The result may be the decreased specificity of an established and generally available antibody (Alexander and Dayal, 1997). Other limitations of microwave heating are listed on pages 142-144. Antigen retrieval can also be accomplished by treating paraffin sections with digestive enzymes. A number of proteolytic enzymes, including trypsin, pepsin, pronase, and ficin (a plant enzyme), have been used for unmasking antigens. However, effectiveness of each enzyme is limited to a few types of antigens. Moreover, enzymatic digestion is ineffective for antigen retrieved in overfixed tissues and tends to damage cell morphology, especially when the treatment is prolonged. Cumulative evidence indicates that heating is generally better than digestion. Therefore, the latter approach is not preferred unless the former is unsuccessful. Better results are obtained in some cases when enzyme digestion is used in conjunction with heat treatment.

Introduction

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Immunohistological diagnosis is critically linked to an assessment of the morphological appearance of the cell. This is accomplished by employing a panel of monoclonal antibodies to establish the immunoprofile of a tumor and, it is hoped, to minimize the risk of false-negative or false-positive staining. Therefore, a thorough knowledge of the chemistry of tissue processing, including antigen retrieval, is essential for routine practice. The antibodies described in this volume are immunoreactive in fixed, paraffin-embedded tissue sections and therefore are the mainstay of routine diagnostic histopathology. However, it should be noted that the specificity of certain monoclonal antibodies, as commonly understood, is not valid because such an antibody shows cross-reactivity, that is, binding to epitopes shared by related proteins in the same tissue or from different tissues. Finally, the use of antibodies to clinically important antigens for diagnostic and prognosis purposes requires a complete understanding of the role of antigens in the biology of disease. For correct interpretation of histological findings, the pathologist must be knowledgeable about pathophysiology, notwithstanding the availability of the most specific panel of monoclonal antibodies. For this and other reasons the role of several antigens, in health and disease is discussed in detail in Chapters 10, 11, and 12. The following discussion summarizes the role of estrogens, p53, Ki-67, PCNA, and p185 proteins in health and disease. The importance of estrogens in health and disease becomes apparent when one considers that these hormones trigger and govern essential functions such as growth, differentiation, and the functioning of many target tissues. They also significantly influence the proliferative and metastatic states of breast cancer cells. It is also known that estrogens affect the regulation of gene transcription through interaction with at least two estrogen receptors ( and ). The role of estrogens and their receptors in breast cancer is emphasized in this volume. As examples, immunohistochemical localization of these receptors in the prostate tissue and in fine-needle aspirates of breast is presented. The tumor suppressor gene p53 encodes a nuclear phosphoprotein which is expressed in most, if not all, tissues of the body. The steady-state levels of this protein in normal somatic cells are usually very small because this newly synthesized protein is highly sensitive to ubiquitin/proteasome-mediated degradation, preventing its accumulation in cells. It functions primarily as a transcription factor that is activated in response to genotoxic stress, including DNA damage. Thus it controls the expression of many genes involved in regulating the cell cycle and apoptosis. In this way, p53 prevents the excessive accumulation of mutations and harmful cells which could give rise to malignancies. On the other hand, mutation of p53 occurs frequently in human oncogenes. These mutations in tumors abrogate the regulatory function of p53 on the cell cycle and lead to increased half-life of the otherwise very unstable wild-type p53 protein. The importance of immunohistochemical localization of p53 protein becomes obvious when considering that approximately 50% of all human malignancies exhibit mutation and aberrant expression of this protein, making it an important target candidate for cancer immunotherapy. Details of immunohistochemical localization of wild-type p53 and mutant p53 are explained in this volume. Ki-67 is a nuclear and nucleolar phosphoprotein. Although the biological function of Ki-67 has not been fully elucidated, it is accepted that this antigen, along with p53, PCNA, cyclin Dl, and bc12, plays an important role in regulating somatic cell proliferation. Immunohistochemical examination using a Ki-67 labeling index is a promising proliferation marker, as a higher rate of Ki-67-positive cells correspond to greater malignancy.

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Monoclonal antibody MIB-1 is used to recognize this antigen, and so the usefulness of this antibody is discussed in detail in Chapter 2. Immunohistochemical methods using microwave heating or autoclave treatment for localizing Ki-67 are presented. Proliferating cell nuclear antigen (PCNA) is an auxiliary protein to DNA polymerase 8 and is intimately associated with DNA replication. Indeed, direct interaction between DNA polymerase and its processivity factor PCNA is essential for effective replication of the eukaryotic genome. This protein also plays a key role in other functions, such as nucleotide excision repair, mismatch repair, base excision repair, cell cycle control, apoptosis, and transcription. These interactions support the concept that PCNA plays a central role in connecting all these important cellular processes and can function as cellular communicator in cells. Clinically useful activity of PCNA can be identified by immunohistochemistry and flow cytometry. Expression of this antigen in a cell population equates to the growth fraction, that is, the proportion of cells involved in an active cell cycle. Because PCNA is expressed in all cycling cells, the entire proportion of dividing cells present at any instant in a population can be detected. Details of immunohistochemical staining of PCNA on cryostat and paraffin-embedded sections are presented in this volume. In recent years evidence has increasingly demonstrated the importance of protooncogenes in the pathogenesis of diseases, including breast cancer. Amplification of HER-2/neu gene is found in ~25% of human breast cancers and results in the overexpression of p185 oncoprotein. Amplification of the gene in breast cancer patients is correlated with shorter disease-free states and poorer overall survival rates than in patients showing no such amplification. Accurate detection of the gene amplification in breast cancer tissues is important in determining patient prognosis as well as response to standard chemotherapeutic agents. Moreover, it is currently the sole criterion for selecting patients for HER-2/neu-targeted therapy with the recombinant humanized anti-p185 antibody Herceptin (trastuzumab) (Pauletti et al., 2000). Overexpression of this protein in breast cancer is associated with adverse prognostic factors that include advanced pathological stage, number of metastatic axillary lymph nodes, absence of estrogen and progesterone receptors, increased S-phase fraction, DNA ploidy, and high nuclear grade. Because the gene product is ultimately responsible for the biological activity of the gene, it is apparent that direct measurement of the protein or immunohistochemical analysis is as clinically relevant as is the determination of the number of gene copies (Battifora et al., 1991). Immunohistochemistry is superior to biochemical assays because it eliminates the dilution effect caused by variable amounts of stroma and other nonneoplastic tissues. Another advantage of immunohistochemistry is its ability to detect overproduction of an oncoprotein resulting from a mechanism other than gene amplification. Amplification of HER-2/neu gene and Overexpression of p185 are also found in many tissues other than those with breast cancer. Immunohistochemical detection of p185 is presented in Chapter 12.

CYTOGENETIC EVALUATION OF TUMORS
A brief discussion on the usefulness of cytogenetic evaluation of tumors is in order. The diagnosis of tumors on the basis of information obtained from immunohistochemical staining alone on occasion may pose a challenge to the pathologist. Cytogenetics, which is another approach to determine the histogenetic origin of some tumors and to identify

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sites of gene deregulation for molecular analysis, can provide an important adjunct to diagnostic surgical pathology. For example, karyotypic analyses are helpful in the differential diagnosis of histologically similar small round cell tumors, including lymphoma and neuroblastoma (Sreekantaiah et al., 1994). These tumors are composed of primitive cells that often lack distinguishing features. Each of these tumors contains specific chromosome changes; thus, cytogenetic analysis provides a reliable approach that can distinguish between these neoplasms. In addition, the identification of diagnostic chromosome translocations in histologically undifferentiated tumors may support a diagnosis that is doubtful on histological grounds alone or may even lead to a reconsideration of the histological diagnosis. This approach can aid in directing therapy, determining prognosis, and identifying sites of gene perturbation for molecular characterization. Unfortunately, cytogenetic evaluation of tumors is still a relatively underutilized approach. However, new potentially promising tumor markers have been introduced based on the molecular genetic cancer research. Various genetic alterations important in carcinogenesis, of which alterations in the ras oncogenes and the p53 tumor suppressor gene are the most common, have now been described. Both of these are useful targets for diagnostic purposes. This is substantiated by considering that p53 alterations are among the most frequent genetic alterations in human malignancies. Similarly, clinical application of ras gene mutation, for example in the diagnosis of pancreatic adenocarcinoma, has been well established (e.g., Berthelemy et al., 1995). Chromosomal abnormalities in many tumors and their diagnostic relevance are discussed by Sreekantaiah et al. (1994) and Gisselsson et al. (2001). Finally, cancer is a genetic disease, for acquired genetic aberrations cause the disease. Changes in antigen expression detected with immunohistochemistry in some instances reflects genetic alterations. The detection of these aberrations at the chromosome and gene levels improved diagnosis, prognosis, and therapy. Therefore, the combination of morphology with genetics is a major step toward a better understanding of human disease. A number of techniques that facilitate this combination are available, such as in situ hybridization, comparative genomic hybridization, expression profiling using array technologies, high throughput screening approaches, and phenotype/genotype correlations on the DNA, RNA, or protein level (Ried et al., 1999). Technological innovations such as image analysis systems, cytophotometric and integrated densitometric quantitation, and computer hard- and software development also assist in this effort.

GENETIC INSTABILITY OF TUMORS
It is well established that cancer is caused by the accumulation of mutations in the genes that are directly responsible for cell birth or cell death. Genetic instabilities are a consequence of cancer mutations. One or more mutations initiate tumor growth, which give the tumorous cell a selective advantage over other cells. The clone derived from the tumorous cell then expands. Successive mutations occur, each followed by waves of clonal expansion. Information on the molecular and physiological bases of genetic instability of tumors is resulting in new approaches to treating cancer. One of two levels of genetic instability correlates with the vast majority of cancers. In most cancers genetic instability is observed at the chromosome level, resulting in losses

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13

or gains of whole chromosomes or large portions of them (Lengauer et al., 1998). On the other hand, in a small subset of tumors, genetic instability is observed at the nucleotide level and results in base substitutions, deletions, or insertions of a few nucleotides. An understanding of these instabilities is providing new insights into tumor pathogenesis. Four major types of genetic alterations that affect growth-controlling genes have been identified in neoplastic cells and are the basis of human cancers.
1. Sequence changes involving base substitutions, deletions, or insertions of a few nucleotides. This type of subtle change is exemplified by missense mutations in the K-ras gene, which occur in more than 80% of pancreatic cancers (Almoguera et al., 1988). These changes cannot be detected with cytogenetic analysis. 2. Alterations in chromosome number involve losses or gains of whole chromosomes and are found in almost all major types of human tumors. Losses of heterozygosity (losses of a maternal or paternal allele) are widespread. The average cancer of the colon, breast, pancreas, or prostate may lose ~25% of its alleles, and some tumors may lose more than half of their alleles (e.g., Vogelstein et al., 1989). Such cancers exhibit a true chromosomal instability that persists throughout the lifetime of the tumor. It is known that chromosome 10 is lost in glioblastomas, inactivating the tumor suppressor gene PTEN (Wang et al., 1997). The gain of chromosome 7 in papillary renal carcinomas indicates a duplication of a mutant MET oncogene (Zhuang et al., 1998). 3. Chromosome translocations are common in certain human cancers. Translocations such as fusions of different chromosomes or of normally noncontiguous segments of a single chromosome can be detected cytogenetically. At the molecular level, such translocations can produce fusions between two different genes, imparting to the fused transcript the tumorigenic properties. For example, in chronic myelogenous leukemias the carboxy terminus of the c-abl gene on chromosome 9 is joined to the amino terminus of the BCR gene on chromosome 22 (Nowell, 1997). Translocation can also cause gains or losses of chromosomal material and generate new gene products. Simple translocations are characterized by distinctive rearrangements of chromosomal segments in specific neoplastic diseases, including leukemias and lymphomas. These specific translocations are necessary for the development and progression of the neoplasms in which they occur. 4. Gene amplification is an important process in human cancers, as it is associated with tumor progression, has prognostic significance, and provides a target for therapeutics (Lengauer et al., 1998); an example is the amplification of HER-2/neu in breast cancers. At the cytogenetic level, gene amplifications can be detected as homogeneously stained regions or double minutes. At the molecular level, multiple copies of an amplicon containing a growth-promoting gene can be detected. The amplification of N-myc oncogene that occurs in about one-third of advanced neuroblastomas is a good example of tumor progression (Seeger et al., 1985).

Almost all solid tumors are genetically unstable. Translocations and gene amplifications add to the chromosomal abnormalities and may reflect additional mechanisms for generating genetic instability that occurs as tumors grow. Genetic instability is the cause of both tumor progression and tumor heterogeneity. As a result, no two tumors are exactly alike and no single tumor is constituted of genetically identical cells. Tumor

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heterogeneity is an obstacle in standardizing the diagnosis, as well as in selecting therapeutic strategies. However, it should be noted that although genetic instability is essential for neoplasia to develop, the instability may provide equally valid therapeutic targets.

TUMOR HETEROGENEITY
Heterogeneity has been reported in a variety of human tumors. Intraindividual heterogeneity is defined as subpopulations of tumor cells found within one tumor. Heterogeneity among different tumors is called interindividual heterogeneity. The detection of heterogeneity at the tumor level is relevant to explanations of clonality differences, metastatic potential of human tumors, and response to therapy under different treatment regimes. The existence of heterogeneity can be explained by genetic instability of malignant progenitor or stem cells. In addition to other influences, heterogeneity might be one individual factor that explains differences in response and outcome of patients under treatment. Heterogeneity at the tumor cell level can be detected by histological, immunohistochemical, molecular genetic and flow cytometric methods, and polymerase chain reaction. Recently, multiparameter flow cytometry was used for detecting tumor cell heterogeneity (Könemann et al., 2000). This immunophenotyping, with its advantages of characterizing simultaneously a variety of different antigens, allows detection of interindividual as well as intraindividual heterogeneity and malignant subpopulations. The method provides the possibility of characterizing solid tumors according to their immunophenotype and DNA content. Molecules that are potentially involved in tumor invasion, metastasis, differentiation/maturation, and cell interactions can be chosen as target antigens. These include adhesion molecules, cell activation antigens, and cytokine and growth factor receptors.

Histological Microdissection
Tissue heterogeneity of histological specimens is well known. Not only neoplastic cells are heterogenous; a tumor may contain a variable admixture of stromal cells, inflammatory infiltrates, endothelial cells, and preexisting tissue. This complexity hinders the study of molecular genetic alterations. Such studies require precise correlation of molecular genetic characteristics to well-defined cell populations. The presence of multiple cell types close to one another in the tumor may limit the precise significance of changes in specific cells. As a result, even sophisticated techniques become less useful when applied to bulk tissue. Study of uniform cell populations is a prerequisite to understanding differential gene expression in tumors. A number of mechanical techniques for microdissection have been developed to isolate cells for analysis from histological sections (Turbett et al., 1996; Youngson et al., 1995; Going and Lamb, 1996; Moskaluk and Kern, 1997; Lee et al., 1988; Zhuang et al., 1995). The most sophisticated technique is laser-assisted and suitable for microdissection of single cells with minimal risk of contamination (Becker et al., 1997). Some of the microdissection methods are satisfactory but also have certain disadvantages; for example, laser-assisted techniques require expensive equipment. To overcome some of the limitations of microdissection methods presently in use, recently a mechanical technique was developed by Harsch et al. (2001). This device

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consists of an ultrasonically oscillating needle and a piezo-driven micropipette for rapid and precise histological microdissection. The oscillating needle is used to fragment the tissue into subcellular particles that are aspirated into the pipette tip. The method can be applied to paraffin sections or unfixed cryostat sections. It allows a sharp demarcation between the dissected area and unwanted tissue that remains intact for further analysis. Individual colonic crypts can be dissected without collecting any adjacent stroma. Thus, the technique is useful for determining gene expression in defined cell populations.

ANTITUMOR VACCINES
Advances in the molecular characterization of human tumors have led to a better understanding of tumor immunology. These advances reveal that cancer cells exhibit specific patterns of gene expression or molecular alterations, compared with normal cellular counterparts, resulting in the production of tumor-associated antigens. A number of such antigens are self-antigens, which allow conceiving and designing of specific vaccines against virtually every solid tumor. Thus, efficient cancer vaccines should be able to neutralize immune tolerance against such antigens. The idea of controlling cancer by stimulating the immune system is not a recent one. In fact, more than a century ago, bacterial extracts were used for stimulating tumor-specific immune responses (Coley, 1893). Subsequently, immunostimulatory cytokines were used against a number of cancers (Marincola et al., 1995). Passive transfer of cytotoxic immune cells (e.g., lymphocytes) was also tested in humans (Yee et al., 1997). Recently, a number of studies indicate that therapeutic vaccines might be useful in restoring immune defenses against cancer. The following discussion summarizes possible uses of cancer vaccines. An important future strategy for cancer immunotherapy is the use of the next generation of antigen-specific cancer vaccines. Until now, most clinical trials have been performed with end-stage cancer patients because data on vaccine-induced immune responses are limited. There are two variations of the development of antitumor vaccination strategies: (1) developing vaccines utilizing whole tumor cells and (2) working on vaccines targeting defined antigens. The advantage of tumor cell–based vaccines is that these in principle comprise all relevant tumor antigens. Consequently, there is no need for prior identification of the tumor antigens to be included in the vaccine. The limitation of this approach is that it is very difficult to understand the therapeutic effect of these vaccines on the disease. In contrast, the use of vaccines comprising defined antigens enables the improvement of vaccine strategies based on empirical findings (Offringa et al., 2000). This approach allows the systematic analysis of vaccine-induced immunity in relation to clinical response. This advantage strongly argues for the usefulness of antigen-specific anticancer vaccines. The protective effect of tumor cell vaccination is thought to involve defined T cell responses. However, only in selected cases does the detection of T cell immunity against defined antigens coincide with clinical response (Sun et al., 1999). For example, vaccination of patients exhibiting residual B cell lymphoma, using the tumor-specific immunoglobulin idiotype as an antigen, was shown to result in sustained molecular remission, accompanied by idiotype-specific T cell and antibody responses (Reichardt et al, 1999). Another positive example demonstrates that a human papillomavirus-specific vaccine can have therapeutic efficacy against benign neoplastic tumors such as genital warts (Lacey et al., 1999).

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Presently, vaccines expressing tumor-associated antigens are being tested in a therapeutic setting. A specific example of a candidate vaccine is Gastrimmune, which is being tested in patients with advanced gastric and pancreatic cancers (Greten and Jaffe, 1999). The aim of such testing is to specifically evoke or restimulate a specific antitumor immune effector response. The likely immune effector mechanisms involved in tumor control/elimination consist of innate immunity, cytotoxic T cells, NK cells, and antibodies. In addition, the activation of specific T helper cells is thought to be important not only to orient and regulate the immune response but also to sustain immune effector mechanisms in vivo (Bonnet et al., 2001). One way to use vaccines against cancer is by preventing infection by pathogens known to predispose to certain cancers. Approximately 16% of the worldwide incidence of cancer can be attributed to infectious pathogens (Ames et al., 1995). The objective is to decrease cancer incidence by using vaccines against the pathogens. A well-known recent example of this approach is the nationwide hepatitis B vaccination program in Taiwan. This resulted in the substantial decline in the incidence of hepatocellular carcinoma in children (Chang et al., 1997). Other cancers caused or facilitated by viruses against which experimental vaccines are available include Burkitt lymphoma, nasopharynx cancer, adult T cell leukemia, cervical carcinoma, B cell gastric lymphoma, and gastric carcinoma. The efficacy of DNA vaccines has also been compared with that of protein subunit vaccines. The use of plasmid DNAs as vaccines has several potential advantages in addition to ease of manipulation and preparation. For example, unlike most protein subunit vaccines, DNA vaccines are potentially able to stimulate both cell-mediated and humoral immunity. On the other hand, both subunit vaccines and DNA vaccines have perceived safety advantages over the use of live virus vaccines. Recently, Nass et al. (2001) have shown that a DNA vaccine expressing a single herpes simplex virus glycoprotein is safer than live virus immunization in immunocompromised animals and that the magnitude of protection in immuncompetent animals against subsequent challenge approaches the strength of protection achieved by sublethal infection. Another recent example of the development of a DNA vaccine is against the HER-2/neu expressing carcinomas. Foy et al. (2001) have utilized an in vivo murine tumor expressing human HER-2/neu for evaluating potential HER-2/neu vaccines consisting of full-length or various subunits of HER-2/neu delivered in protein or plasmid DNA form. This study demonstrates that protective immunity against HER-2/neu–expressing tumor challenge can be achieved by these vaccines. Partial protective immunity is also observed following vaccination with the intracellular domain (ICD), but not extracellular domain (ECD), protein subunit of HER-2/neu. The mechanism of protection elicited by plasmid DNA vaccination is thought to be exclusively CD4-dependent, whereas the protection with ICD protein vaccination requires both CD4 and CD8 T cells. These early studies indicate that multiple forms of HER-2/neu vaccines would be more effective in eliciting the protective HER-2/neu–specific antitumor responses. It is expected that in the near future antigen-specific vaccines will be applied effectively to induce strong T cell immune responses in patients displaying less progressed stages of disease. A comprehensive discussion on the development of therapeutic cancer vaccines (molecular vaccines) has been presented by Moingeon (2001) and MonzaviKarbassi and Kieber-Emmons (2001).

Introduction

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MOLECULAR GENETICS
Basic mechanisms of pathogenesis can be elucidated through molecular genetic research. Based on such information, specificity, sensitivity, and efficiency of diagnostic and prognostic tests for many diseases can be improved. As a result, new insights into therapeutic approaches can be developed, and their effectiveness can be assessed more reliably. The Human Genome Project has focused attention on the association of mutations in nuclear DNA with human diseases. A complete working draft of the human DNA sequence was completed in spring 2000. This project continues to define disease-associated mutations, and the number of clinically useful molecular pathological techniques and assays has also been increasing. The technology is available to extract and amplify DNA from minuscule archival and fresh samples as diverse as blood, urine, sputum, and solid tissues, including fixed and paraffin-embedded tissues. Also, DNA can be purified even from dried blood spots for amplification and mutation analysis (Kiechle, 1999), thus permitting the study of an individual’s inherited genes and mutations. Hereditary information can also be obtained by assessing RNA, proteins, and enzyme activities. Such assessments can be morphological, immunological, or biochemical. The following examples indicate the usefulness of molecular pathology in better understanding diseases. The usefulness of identifying molecular alterations underlying neoplasia is obvious in borderline tumors. Whether such tumors should be classified as benign or malignant, and whether they represent a precursor of frank malignancy, is a matter of controversy despite extensive clinical and pathological studies. In many cases this problem can be solved by using the biology of tumors as the genetic indicator of malignancy. This approach is exemplified by mutations in the p53 tumor suppressor gene and Ki-ras oncogene, which are the most common genetic alterations in human cancers. These mutations are used as genetic indicators of malignancy. Methods are available to analyze abnormalities in these genes using paraffin sections of neoplasms (Frank et al., 1994; Caduff et al., 1995). Mutations in codon 12 of Ki-ras can be identified in DNA extracted from paraffin sections using an amplified created restriction site method, followed by confirmation using gene sequencing (Lin et al., 1993). Missense mutations in the p53 gene can be identified with an immunohistochemical surrogate to detect the nuclear accumulation of p53 protein that results from such mutations (Kerns et al., 1992). Recently, Caduff et al. (1999) have evaluated abnormalities in p53 and Ki-ras in malignant and borderline ovarian tumors of various histological types in paraffin-embedded tissues. The patterns of these genetic alterations in borderline and malignant neoplasms were compared and correlated with cell type and stage. This preliminary molecular analysis suggests that serous borderline tumors have the same molecular features usually associated with malignancy but are unlikely to represent a precursor of invasive serous carcinoma. On the other hand, mucinous borderline tumors may represent a precursor or variant of mucinous carcinoma of the ovary. Another example is the study of microsatellites that are short DNA loci having simple sequence repeats that are widely distributed throughout the human genome. They are a valuable source for human genetic linkage analysis and molecular cancer research

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because of allelic polymorphisms. In hereditary nonpolyposis colorectal cancer, an autosomal-dominant disorder accounting for 2–10% of all colorectal cancers, length alterations in single mononucleotide or dinucleotide repeats (microsatellite instability [MSI]) occur (Raedle et al., 1999). The MSI is used as a diagnostic criterion of replication errors caused by various mutations in at least five mismatch repair genes (Lynch and Smyrk, 1996). Therefore, MSI analysis is useful in clinical practice to identify patients with hereditary nonpolyposis colorectal cancer. Raedle et al. (1999) have presented a rapid DNA extraction method (rapid microsatellite analysis) for analyzing replication errors in paraffin-embedded tissues. Southern blotting and polymerase chain reaction are being used for detecting B- and T-cell clonality in lymphoproliferative diseases, including mantle cell lymphoma and lymphoma of the breast (Medeiros and Carr, 1999). Molecular genetic tests are currently important ancillary tools for the diagnosis and classification of malignancy, and their role is likely to increase in the future. The positive aspects of molecular genetics and molecular pathology mentioned above need to be balanced with ethical concerns to safeguard the rights and welfare of human subjects (Sobel, 1999). The state and federal regulations protecting patient’s privacy and welfare must be observed.

cDNA MICROARRAY TECHNOLOGY
In conjunction with detailed understanding of the human genome, sophisticated methods are required for gene expression analysis and gene discovery. These approaches will provide insights into growth, development, differentiation, homeostasis, aging, and disease onset. One such recently introduced method is cDNA microarray or DNA-chip technology, which facilitates monitoring the expression of hundreds and thousands of genes simultaneously and provides a format for identifying genes as well as alterations in their activity (Kononen et al., 1998). Because of the wide spectrum of genes and endogenous mediators involved, this technology is helpful in recognizing chronic diseases. As the cDNA microarray technique allows large-scale expression analysis, it is well suited to observe the broad effects of oncogenic transcription factors on gene expression and potentially clarify their role in oncogenesis. The cDNA technology uses cDNA sequences or cDNA inserts of a library for polymerase chain reaction (PCR) amplification, which are arrayed on a glass slide with highspeed robotics at a density of 1,000 cDNA sequences per square centimeter. In other words, microarrays can be constructed from specific cDNA clones of interest, a cDNA library, or a selected number of open reading frames from a genome sequencing database to allow a large-scale functional analysis of expressed sequences. These microarrays serve as gene targets for hybridization to cDNA probes prepared from RNA samples of cells and tissues. A two-color fluorescence labeling technique can be used to prepare the cDNA probes so that a simultaneous hybridization, but separate detection, of signals provides the comparative analysis and the relative abundance of specific genes expressed. The cDNA technology is essentially an array-based, high-throughput protocol that determines gene expression and copy number survey of very large numbers of tumors. As many as 1,000 cylindrical tissue biopsies from individual tumors can be distributed in a single tissue microarray. Sections of the microarray also provide targets for parallel in situ

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detection of DNA, RNA, and protein targets in each specimen on the array. Moreover, consecutive sections allow rapid analysis of hundreds of molecular markers in the same set of specimens. Another advantage is that sufficient cDNA for hybridization to a microarray can be produced from as little as 1 mg of tissue. This technology can be used to profile complex diseases and discover novel diseaserelated genes. It can dissect complex human diseases by analyzing the pattern of gene expression. The cDNA microarray method could provide new targets for drug development and disease therapies and thus facilitate improved treatment of chronic diseases that are challenging because of their complexity. Listed below are examples of diseases whose molecular characteristics have been determined using gene arrays. Ljubimova et al. (2001) have used 11,000 gene microarrays for identifying gene expression profiles in brain tumors, including high-grade gliomas (glioblastoma multiforme [GBM] and anaplastic astrocytoma), low-grade astrocytomas, and benign extraaxial brain tumors (meningioma), and then compared them with normal brain tissue. In this study the gene array method was combined with reverse transcriptase (RT)-PCR and immunohistochemical evaluation of glial tumors. All GBMs overexpressed 14 known genes, whereas these genes were barely detectable in normal human brain tissue. This study also showed that laminin-80–containing GBMs recurred significantly sooner after surgical removal than did GBMs with a predominant expression of laminin-9. Thus, overexpression of laminin-8 in tumor blood vessel walls may be an indicator of time to recurrence for patients with GBM. Another example of the involvement of many different genes in a cancer is renal cell carcinoma. This cancer is one of the 10 most frequent malignancies in western countries. Genes involved in the initiation and progression of this cancer include the von Hippel–Lindau gene on chromosome 3p, the epidermal growth factor receptor gene on chromosome 7p, the transforming growth factor gene on chromosome 2p, and the c-myc oncogene on chromosome 8q (Siezinger et al., 1988; Moch et al., 1998; Lager et al., 1994; Yao et al., 1998). Other genes involved in renal cancer are currently not known. Moch et al. (1999) have combined tumor arrays and cDNA arrays for rapid identification of genes and their role in renal cell carcinoma. They constructed a kidney cancer tissue array consisting of 532 renal tumors, 386 of which had clinical follow-up data available. There were 89 differentially expressed genes in the cancer cell line CRL-1933, one of them encoding for vimentin. Vimentin expression was significantly associated with poor patient prognosis independent of grade or stage. cDNA microarray technology has also been used for verifying the involvement of a number of genes in another complex disease, rheumatoid arthritis (Heller et al., 1997). In this disease inflammation of the joint is caused by the gene products of many different cell types present in the synovium and cartilage tissues plus those infiltrating from the circulating blood. In this study the presence of gene products, such as matrix degrading metalloproteinase (MMP), macrophage inflammatory protein (MIP), and human matrix metalloelastase (HME), was verified. The expression profiles of the genes demonstrate the utility of the microarrays in determining the hierarachy of signaling events. The downstream effects of both PAX3 and PAX3-FKHR on NIH 3T3 cells with cDNA microarrays has also been monitored (Khan et al., 1999). This study elucidated the pattern of gene expression induced by these two oncogenic transcription factors in these cells; these factors showed significant myogenic properties. Other recent examples of the application of gene arrays for determining the molecular parameters of individual tumors

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are ovarian and cervical cancers and metastatic versus primary breast cancer (Ono et al., 2000; Shim et al., 1998; Nacht et al., 1999). New subclasses of leukemia have also been identified using gene arrays, which have become critical for the successful treatment of patients (Golub et al., 1999). Because the individual arrayed tissue samples are very small (0.6 mm in diameter), one might ask if these specimens are representative of their donor tumors! To answer this question, Nocito et al. (2001) studied a set of 2,317 bladder tumors that had been previously analyzed for histological grade and Ki-67 labeling index. The histological grade and the Ki-67 labeling index were determined for every arrayed tumor sample. The grade and Ki-67 information obtained on minute arrayed samples were highly similar to the data obtained on large sections. On the basis of this evidence, it can be stated that intratumor heterogeneity does not significantly affect the ability to detect clinicopathological correlations on the tissue microarrays. It is concluded that tissue microarray is an important tool for rapid identification of biological or clinically significant molecular alterations in tumors.

ANGIOGENESIS
Two distinct processes, vasculogenesis and angiogenesis, form blood vessels. Vasculogenesis is responsible for the de novo differentiation of endothelial cells from mesodermal precursors and occurs during embryonic development, leading to the formation of a primary vascular plexus. Angiogenesis, on the other hand, is the process of sprouting and configuring new blood vessels from preexisting ones. It is a complex phenomenon comprising a series of cellular events that lead to the neovascularization associated with the process of tumor growth, metastasis, inflammation, and wound healing (Fig. 1.5/Plate 1A). Angiogenesis that occurs in wound repair and formation of collateral blood vessels following an infarct, ischemia, or reduced blood flow is advantageous for

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normal tissue function. It should be noted that tumor angiogenesis is not sufficient to cause tumor spread and patient death. Tumor cells must also proliferate, penetrate host tissues and vessels, survive within the vasculature, escape the host immune system, and then begin growth at a new body site (Weidner, 1998). Before discussing the role of angiogenesis in disease, it is relevant to explain the process of angiogenesis. The complex process of angiogenesis includes the recruitment of nearby endothelial cells, their activation, degradation of the vascular basement membrane, proliferation and form a new capillary (Albini et al., 2000). Tumor-induced endothelial cell activation leads to the acquisition of a phenotype characterized by chemotactic motility, basement membrane invasion, and proliferation. These events are followed by differentiation into a new vessel. During the last decade there have been significant advances in the understanding of functional mechanisms of the molecules involved in angiogenesis. Angiogenesis is mediated by multiple positive and negative regulator molecules released by tumor cells, intratumoral macrophages, mast cells, and endothelial cells. The balance of the effects of these mediators determines the outcome of this process. At least three groups of extracellular signals are involved in angiogenesis: (1) soluble growth molecules such as acid and basic fibroblast growth factors and vascular endothelial growth factor (discussed later) that affect endothelial cell growth and differentiation; (2) factors such as transforming growth factor and angiogenin that inhibit proliferation and enhance differentiation of endothelial cells; (3) extracellular matrix–bound cytokines released by proteolysis, which contribute to angiogenic regulation. Other growth factors implicated in different steps of angiogenesis are platelet-derived growth factor, hepatocyte growth factor, and angiopoietins 1 and 2. Also, various endothelial surface molecules, such as CD31, CD144, and integrins, play a role in angiogenesis. Some of the above-mentioned secreted factors are angiogenic, whereas others are angiostatic. Thus, angiogenesis is mediated by multiple positive and negative regulatory molecules released by both tumor cells and the surrounding normal cells. The balance between these regulators determines whether or not neovascularization will occur. Indeed, antiangiogenic therapy is based on the use of negative regulators of neovascularization aimed at suppressing the proangiogenic signal or increasing the inhibitory signals. Albini et al. (2000) have used the gene therapy approach using class I interferons for effectively inhibiting tumor angiogenesis and growth of vascular tumors. Although overwhelming evidence indicates that endothelial cells are central to the angiogenic process, the following discussion proposes the role of tumors in the formation of blood vessels. According to Maniotis et al. (1999) and Folberg et al. (2000), blood vessels of malignant eye tumors known as uveal melanomas are formed by tumor cells instead of endothelial cells. These highly aggressive and metastatic cells are capable of forming in vivo and in vitro vascular channels, which consist of a basement membrane that stains positive with the periodic acid–Schiff (PAS) reagent in the absence of endothelial cells and fibroblasts. The generation of such channels is termed vasculogenic mimicry. This evidence suggests that angiogenesis may not be the only mechanism responsible for creating tumor microcirculation. Therefore, methods used to identify the tumor microcirculation by staining endothelial cells may not be applicable to tumors that express vasculogenic mimicry. However, certain aspects of the concept of vasculogenic mimicry have been questioned by McDonald et al. (2000). They indicate that PAS-stained channels do not

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represent the microvascular architecture, and endothelial cell–lined blood vessels are also present in uveal melanomas. They moreover report that tumor cell-lined vessels are infrequent in these melanomas. Nevertheless, tumor cells can acquire a new phenotype and participate in the formation of blood vessels. It is concluded that the extent and the pathophysiological significance of cancer cells becoming lining cells and participating in the formation of blood vessels in tumor is still unclear. Information on the angiogenesis regulatory molecules has produced new therapeutic strategies for suppressing angiogenesis and tumor growth or promoting angiogenesis against coronary and peripheral ischemia and stimulation of wound healing (Thompson et al., 1999; Kahn et al., 2000). Limited space does not allow discussion of these aspects of angiogenesis, except for VEGF, which is the most potent angiogenic factor (see pages 23–24). Angiogenesis plays an important role in the development and progression of a number of disease states, including various cancers, diabetic retinopathy, macular degeneration, psoriasis, and rheumatic arthritis. The tumor microcirculation plays a key role in hematogenous dissemination of cancers. There is compelling evidence that angiogenesis is indeed critical for tumor growth progression and metastasis because tumors require new blood vessels to achieve a size larger than 2–3 mm. Also, a considerable amount of evidence suggests that tumor angiogenesis is crucial for the growth of solid tumors in vivo (Folkman, 1996). The growth of tumors (including solid tumors) must be preceded by an increase in capillaries and newly formed blood vessels that provide tumor cells with oxygen and nutrients as well as paracrine mediators. The blood vessels also remove waste products. A large number of tumor blood vessels increases the opportunity of the tumor cells to enter the circulation. In fact, the newly formed capillaries usually have a fragmented basement membrane, facilitating easier invasion. In the prevascular phase, with little or no angiogenic activity, the tumor is unable to expand beyond a few cubic millimeters, but once angiogenic factors are released in sufficient number, the onset of angiogenic activity stimulates rapid expansion of the tumor. The microvessel density of the tumor mass, a measure of tumor angiogenesis, correlates with metastasis and can be used as an independent prognostic factor in the management of cancer (Jacquemier et al., 1998). A number of studies indicate that microvessel density gives prognostic information on breast cancer (Weidner et al., 1993). With respect to prognostic carcinoma, it is thought that a low vascular density correlates with significantly longer survival duration than with carcinomas having high vascular density. Thus, neovascularization has proven to be an independent predictor of pathological state in prostatic carcinoma. A correlation between the endocrine differentiation and increased neovascularization in prostatic cancer has also been reported (Grobholz et al., 2000). High-grade tumors with a high neuroendocrine differentiation and increased neovascularization indicate high risk and unfavorable outcome. Although the role of angiogenesis as a prognostic factor has been most widely analyzed in breast cancer, angiogenesis also plays an important prognostic role in other carcinomas such as gastric cancer. This cancer is a highly aggressive malignancy with poor prognosis and low survival rates. Sanz-Ortega et al. (2000) have evaluated advanced gastric cancers for the expression of oncogenes HER-2/neu (c-erbB-2), c-myc, and epidermal growth factor receptor, as well as microvessel density. Avidin-biotin immunohistochemistry using CD34 stained paraffin sections has shown that tumor angiogenesis is the most important independent prognostic indicator to predict overall survival.

Introduction

23

Immunohistochemistry using primary antibody against rabbit antihuman VEGF (diluted 1:1000) has demonstrated that angiogenesis is also a vital process in cartilaginous tumors and that VEGF expression by malignant chondrocytes is required for the formation of intracartilage vessels (Ayala et al., 2000). Intracartilage vessels might be involved in the acquisition of metastatic potential by cartilage tumors. Squamous cell carcinoma is also characterized by a richly vascularized stroma and overexpression of VEGF. This carcinoma of the skin is a malignant tumor of epidermal keratinocytes with a destructive growth pattern, and it has the ability to metastasize. It has been demonstrated that selective overexpression of VEGF in highly differentiated squamous cell carcinomas is sufficient to induce tumor invasiveness as well as to promote tumor growth and angiogenesis (Detmar et al., 2000). The tumor stroma also plays an active role in the progression of this cancer. As stated earlier, angiogenesis plays a role in repairing blood vessel injury. Two systems, angiogenesis and hemostasis, remain poised for repair of blood vessel injury. At the site of blood vessel injury, adhered platelets secrete both positive and negative regulators of angiogenesis, mainly from internal The positive regulators include VEGF; negative regulators include platelet factor 4. Hepatocyte growth factor affects both stimulation and suppression of angiogenesis. On the other hand, the hemostatic system maintains the liquid flow of blood by regulating platelet adherence and fibrin deposition. Browder et al. (2000) have discussed in detail how angiogenesis is coordinated by and with hemostasis during blood vessel repair. In conclusion, sufficient evidence is available indicating that assessment of microvessel density is very useful in tumor biology. However, consensus on the prognostic value of angiogenesis is lacking. The main reasons for conflicting results consist of the study of different angiogenic-regulating factors, the use of varying methodologies for measuring microvessel density, and the significant intraobserver variation that exists in interpretation of the number of positive vessels and the optimal way in which fields are selected, that is, hot spot (the most vascular area of the tumor) versus general counting of vessels. These reasons do not allow meaningful comparison among the results reported in various studies. Standardization of processing conditions, such as tissue section preparation, staining, careful selection of the hot spot, and a strict protocol for defining microvessels, can achieve adequate reproducibility. However, despite these precautions, manual counting of microvessels and selection of the hot spot are still subjective and therefore not always fully reproducible. These problems can be significantly minimized by using fully automated microvessel counting and hot spot selection by image processing of whole tumor sections, for example, in invasive breast cancer (Beliën et al., 1999). In comparison with the manual method, the automated procedure reduces the microvessel measurement time when the complete tumor is scanned, achieves greater accuracy and objectivity of hot spot selection, and allows visual inspection and relocation of each measurement field awards. The reasons for contradictory interpretations and potential remedies have been presented in more detail by Hansen et al. (1998).

Vascular Endothelial Growth Factor
The vascular endothelial growth factor (VEGF) is a member of the six-member VEGF family: VEGF, placenta growth factor, VEGF-B, VEGF-C, VEGF-D, and VEGF-E. These

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members have overlapping but specific roles in the growth of new blood vessels. The following discussion is limited to only one member, VEGF (also known as vascular permeability factor), which is the most important and most frequently studied angiogenic factor. It is a homodimeric 34–42 kDa glycosylated heparin-binding glycoprotein. Alternative exon splicing of the VEGF gene produces multiple species of mRNA, which encode different VEGF protein isoforms having subunit polypeptides of 121, 145, 165, 189, or 206 amino acid residues (Neufeld et al., 1999). Vascular endothelial growth factor has three receptors [VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), and VEGFR-3 (FLT-4)], each consisting of seven immunoglobulin-homology domains, a transmembrane sequence, and an intracellular portion containing a split kinase domain (Shibuya, 1995). Ligand (VEGF) binding induces receptor dimerization and subsequent auto/transphosphorylation. The receptors have distinct roles in vasculogenesis and angiogenesis during embryonic development. Precise roles of the three receptors have been discussed by Veikkola and Alitalo (1999). Transcription of VEGF mRNA is induced by a variety of growth factors and cytokines, including platelet-derived growth factor-BB, epidermal growth factor, tumor necrosis transforming growth and (Ferrara and Davis-Smyth, 1997). Tissue oxygen tension tightly regulates VEGF levels, and exposure to hypoxia rapidly and reversibly induces VEGF expression through both increased transcription and stabilization of the mRNA (Levy et al., 1996). Hypoxic upregulation of VEGF thus provides a compensatory mechanism by which tissues can increase their oxygenation through induction of blood vessel growth. Normoxia downregulates VEGF production and leads to regression of certain newly formed blood vessels. By these opposing processes the vasculature becomes matched to the tissue oxygen demands (Veikkola and Alitalo, 1999). The vascular endothelial growth factor is produced by tumor cells, macrophages, and endothelial and smooth muscle cells. It induces vascular endothelial cell migration, enhances vascular permeability, and promotes extravasation of plasma proteins from tumor vessels to form an extracellular matrix, facilitating inward migration of endothelial cells (Callagy et al., 2000). These characteristics impart selectivity to VEGF for endothelial cells. The vascular endothelial growth factor is involved in angiogenesis in a wide variety of biological systems, including the female reproductive cycle, wound healing, and tissue repair. Proliferation of blood vessels during the formation of the corpus luteum in the ovary and during the growth of endometrial vessels in the uterus occurs upon expression of the VEGF mRNA and protein (Ferrara and Davis-Smyth, 1997). This factor is also detected during angiogenesis occurring at the site of embryo implantation in the uterus (Shweiki et al., 1993). In ischemic cardiac tissue, VEGF mRNA is increased, suggesting the involvement of this factor in the growth of collateral blood vessels (Hashimoto et al., 1994). In addition to its role in physiological angiogenesis, VEGF is active in pathological neovascularization. For example, squamous cell carcinoma of the skin strongly expresses VEGF (Weninger et al., 1996). In fact, tumoral VEGF correlates with prognosis in a variety of tumors, including breast cancer and malignant mesothelioma (Fig. 1.6).

Immunohistochemical Localization of Vascular Endothelial Growth Factor
The following method is recommended for immunostaining vascular endothelial growth factor (VEGF) (Callagy et al., 2000). Breast cancer tissues (including the invasive

Introduction

25

edge of the tumor) are fixed with formalin and embedded in paraffin. Sections thick) are mounted onto adhesive-coated slides, dried, and then deparaffinized. The sections are treated with 3% hydrogen peroxide for 5 min to block endogenous peroxidase activity. After washing in PBS, the sections are placed in 10 mM sodium citrate buffer (pH 6.0) and boiled for 5 min in a microwave oven to unmask the antigens. They are washed in Tris buffer sodium chloride (25 mM Tris-HCl [pH 7.6] and 150 nM sodium chloride) and incubated in normal goat serum (diluted 1:10 with TBS) for 30 min to block nonspecific staining. The sections are incubated in the rabbit polyclonal anti-VEGF (Santa Biotechnology, CA) and diluted 1:100 for 30 min at room temperature. Antigen-antibody reaction is detected using the biotin-streptavidin–based detection kit (Dako). The reaction is developed using DAB+hydrogen peroxide and counterstained with Mayer’s hematoxylin. Exclusion of the primary antibody serves as a negative control. The results of this procedure are shown in Fig. 1.7. Telepathology (Telemedicine) Telepathology, introduced by Weinstein et al. (1987), is a pathology practice that requires telecommunication technologies to transmit digital images to distinct sites for diagnostic, consultation, and educational purposes. Telepathology is an affordable option in places where a pathologist is unaffordable, such as rural hospitals that are too small to support a pathologist. In addition, telepathology facilitates seeking a second opinion on a difficult case. It is thought to be accurate and cost-effective, and its advantages outweigh the problem of waiting longer to have a slide read. Also, the resolution obtainable with videomicroscopy is thought to be adequate and appropriate for diagnosis. Telepathology is expected to become an integral part of medical practice for practical, economic, and humane reasons.

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Telepathology can be divided into two major modalities: static imagery and dynamic (real-time) imagery. Each of these two methods has advantages and limitations. Static imagery (the store-and-forward method) involves capturing of still images from a microscope and transmitting them through a point-to-point connection or by transmission control protocol/internet protocol. The still digital images selected at the remote site are transmitted at a later time for remote diagnosis. The images provided are of superior quality, but the number of images is limited. It is usually not feasible to transmit the images in real time, and the selection of images by the remote site requires two pathologists to share the interaction. The images can be transmitted over the Internet because bandwidth (role of data transmission) requirements are low for the static imagery. In static imagery, expedient delivery of high-resolution images can be achieved by attaching pathology images with an electronic mail message. This method is applicable when real-time consultation is not required. Despite the low cost and simplicity of static imagery,

Introduction

27

it has certain shortcomings. Because static imagery uses relatively low-resolution digital photomicrography, it requires high optical magnification to allow adequate examination of diagnostic images. This results in the need to collect and transmit multiple image files. Furthermore, static image acquisition means that fields for imaging are preselected by a person other than the telepathology consultant, leading to unattended field selection biopsy error (Weinstein et al., 1997). However, high-resolution digital scanning cameras allow the acquisition of digital images up to 3,400 × 2,700 pixels of resolution. These images can be captured at a relatively low optical magnification and digitally magnified multiple times without visible degradation. They can be scrolled at different magnifications in computer, simulating light microscopy. This high-resolution digital photomicrography and the Internet have been used for telepathological gastrointestinal biopsy consultations (Singson et al., 1999). In contrast to static imagery, in dynamic imagery the consultant examines a histological or cytological slide from a remote site by using sophisticated robotic microscopes that transmit real-time digital images through fast and expensive telecommunication links that provide very high band widths. According to Weinstein (1996), low-resolution dynamic images are more useful to a pathologist than high-resolution static images. The diagnostic accuracies for static imaging and dynamic imaging are 88% and 96–97%, respectively. The latter range falls within the acceptable range for surgical pathology. Although the real-time telepathology shows a higher diagnostic accuracy, static imagery continues to be the dominant method used. Static imaging is adequate in those cases where tissue sampling is not a problem. Attempts have been made to develop systems that combine the advantages of static imagery and dynamic imagery. Such systems have been described by O’Brien et al. (1998). However, few of these systems have been implemented because of their complexity. Recently, a new hybrid telepathology system has been described, which achieves dynamic real-time microscopic video transmission for providing dynamic imaging. The implementation of this system is awaited. Imaging standards remain an issue in telepathology. Lack of critical literature in this field is also a barrier to further development and acceptance of this technology. Furthermore, standards must be developed and accepted for the types of cases that will be diagnosed and for protecting patients’ privacy. In addition, telepathology equipment is more expensive than teleradiology equipment. The most extensive and well-known telepathology service is part of the U.S. Armed Forces Institute of Pathology. This service offers the diagnostic evaluation of microscopic still images sent via e-mail (http://www.afip.org/). Recently, telemicroscopy via Internet browsers such as Netscape Navigator and Internet Explorer was introduced by Wolf et al. (1998a, b). They reported a new concept in Internet functionality by demonstrating how Internet browsers with Java support can use remote control of computer-controlled devices such as an automatic microscope. More recently, Petersen et al. (2000) reported how this technology can be used for image transfer and communication between pathologists or research scientists. Essentially, it is based on a conventional light microscope with a video camera, which in turn is connected to a computer with a frame grabber and Internet access. This Telemic system allows the user to show and discuss microscope images with any pathologist who is connected to the Internet. For inquiries about the software and information on the installation, the reader should contact the Telemic homepage at http://amba.charite.de/telemic

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FUTURE OF IMMUNOHISTOPATHOLOGY
An increasing understanding of the molecular changes associated with various tumor groups, and the genetic variability within them, is beginning to provide important new information about clinical progression and prognosis (Graadt van Roggen et al., 1999). Many malignant tumors carry chromosomal aberrations detectable at a cytogenetic or molecular level. Some of these changes are nonrandom and associated with specific tumor types (Ladanyi, 1995). Chromosomal analysis aimed at detecting specific alterations is the most effective variable in resolving the frequent diagnostic dilemmas. The detection and description of apparent tumor-specific genetic alterations within the sarcoma group are already beginning to play an increasingly important role in unraveling and understanding the molecular biology of tumorigenesis. For example, mutations in the retinoblastoma gene (Rb) have been detected in a large proportion of high-grade sarcomas (Sreeckantaiah et al., 1994). Also, the coinactivation of p53 and Rb indicates that both genes may be involved in tumorigenesis of certain sarcomas. In fact, the identification of increasing numbers of tumor-specific genetic alterations has become an helpful adjunct to histopathological assessment in reaching a correct diagnosis. The above observations suggest that an accurate histological classification of tumor types is useful in establishing meaningful clinical trials of optimal management strategies. The relationship between immunohistopathology and surgical pathology becomes apparent when one considers that a large number of monoclonal antibodies is being produced that detect cells at each stage of cancer development. These antibodies are directed against antigens that determine levels of proliferation, angiogenesis, proteolysis, and cell adhesion (Elias, 1999). Thus, it has become possible to determine biochemical alterations occurring during the cell’s progression to malignancy. In addition, new oncogenes are being discovered at a rapid pace. These developments are helping pathologists and oncologists to refine therapeutic and prognostic decisions. Furthermore, these advancements, along with increasing understanding of the molecular and cellular mechanisms of cancer, are expected to lead us to the evaluation of a person’s risk of developing cancer. The ultimate goal, of course, is to prevent cancer. The relationship between gene expression profiles and cellular behavior in humans is largely unknown, and expression patterns of individual cell types have yet to be precisely measured (Emmert-Buck et al., 2000). Although we know that the human genome consists of 32,000 genes, at present the function of only a relatively small percentage of genes is known. However, it is hoped that our understanding of how gene expression modulates cellular phenotype and response to the environment will be achieved within the next few years or a few decades. In June 2000, the International Human Genome Project and Celera Genomics Corporation announced the completion of a “working draft” of the human genome sequence, the genetic code that carries the instructions allowing us to develop, grow, and live. It is possible now to understand the secrets of life processes to an extraordinary degree, to personalize medicine and offer clues to the differences and remarkable similarities among us. Human genome information in concert with full-length cDNA sequencing of all genes will also lead us to an exciting new paradigm in biomedical research known as molecular profiling (Emmert-Buck et al., 2000). Molecular profiling will facilitate the identification of individual genes and collection of genes that mediate particular aspects of

Introduction

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cellular physiology and pathology, thus improving our understanding and treatment of diseases. I am confident that with the approach of the postgenome era, an ever-increasing number of human genes will be discovered and their functions elucidated. Combined with the knowledge of human gene polymorphism, genotyping will allow prediction of the genetic predisposition to certain diseases, such as cancer. The new millennium will usher us in a new era of disease-predictive medicine.

PREPARATION OF BUFFERS
Tris-buffered saline (TBS) (Stock solution)
Tris NaCl Distilled water Adjust pH to 7.8 Distilled water to make 5 liter 303 g 450 g 4 liter 185 ml HCl (32%)

Dilute 10 times with distilled water before use

Phosphate-buffered saline (PBS) (Stock solution)
Disodium hydrogen phosphate dihydrate Potassium dihydrogen phosphate NaCl Distilled water Adjust pH to 7.4 Distilled water to make 5 liter 70.5 g 10.5 g 450 g 4 liter

Dilute 10 times with distilled water before use

Citrate buffer for heat-induced antigen retrieval
Citric acid monohydrate 2.1 g Distilled water 4 liter Adjust pH to 6.0 with 13 ml 2 N NaOH solution

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Chapter 2

Antigens and Antibodies

It is instructive to define relative terminologies. An antigen is a molecule that combines with a specific antibody but which itself may not necessarily be immunogenic. An immunogen is a cell or macromolecule that stimulates a specific immune response. An epitope (an antigenic determinant) is the site on a complex antigenic molecule which is recognized by the antibody. An antibody (immunoglobulin) is a glycoprotein molecule produced by differentiated B lymphocytes when stimulated by an antigen. Immunoglobulin G (IgG) is the most abundant class of immunoglobulins in human serum. IgG is the primary Ig molecule produced during the secondary immune reaction to the antigen. Immunization is the administration of an antigen to an animal to evoke the production of antibodies. Serum is the blood plasma from which the fibrogen has been removed. Mammalian sera contain ~8% (w/v) protein, consisting of approximately equal proportions of albumin and globulin. Antiserum is the serum containing antibodies to an antigen. Fab is the fragment of an immunoglobulin (Ig) that binds to an antigen and is produced by treating the Ig molecule with the enzyme papain. is the fragment of an Ig molecule which contains both antigen-binding sites and the disulfide bridge. It is produced by treating the Ig molecule with the enzyme. Fc fragment is the part of an Ig molecule that has no antigen-binding activity but binds to FC receptors on phagocytes and may activate complement. A clone is a group of daughter cells that are produced from a single cell. Hybridoma is the cloned hybrid cells formed by the fusion of an antibody-forming cell and a malignant myeloma cell. A hybridoma grows continuously and produces antibodies of a single specificity termed monoclonal antibodies. Affinity is the association constant at the equilibrium between an epitope and a single antigen-binding site of the antibody, independent of the number of sites. This term describes the strength and the stability of the binding. Specificity refers to the selective binding between an antigen and its corresponding antibody. Titer is the measure of units of antibody per unit volume of serum. The concentration of the antibody is determined by titration.

ANTIGENS
An antigen is a substance that reacts specifically with receptors on the surface of lymphocytes and with their soluble products such as antibodies. Antigens usually are large, complex protein or polysaccharide molecules with molecular weights usually greater than
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40,000. However, the molecular weight, for example, may vary from 15,000 (hen egg white lysozyme) to 2,000,000 (keyhole limpet hemocyanin) daltons. Protein antigens function as the most potent immunogens, and polysaccharide antigens rank second. For cell-mediated immunity, only proteins serve as immunogens. Certain nucleic acid types such as Z-DNA and other molecules can also stimulate antibody production. Antigens can be defined on the basis of four immunological properties: immunogenecity, antigenicity, allerogenicity, and tolerogenicity. The ability of a substance to induce an immune response is called immunogenicity. Most antigens have a variety of different antigenic determinants (epitopes) on their surfaces, which stimulate antibody production. Antigenicity is the ability of an immunogen to combine with an antibody or cell surface receptors. Allerogenicity is the ability to induce various types of allergic responses. Allergens are immunogens that tend to activate specific types of humoral or cell-mediated responses having allergic manifestations (Kuby, 1992). Tolerogenicity is the capacity to induce specific immunological nonresponsiveness in either the humoral or the cell-mediated systems. In other words, experimentally induced tolerance can be defined as a state in which an animal fails to respond to an antigen that would normally be immunogenic. It is not known whether similar mechanisms generate both naturally acquired self-tolerance and experimentally induced tolerance. Immunogens induce an immune response only if they are recognized as foreign (nonself). A case in point is protein bovine serum albumin, which is immunogenic in sheep but not in cows. Most large antigens have multiple reactive sites, or epitopes, on their surfaces, which can induce production of specific antibodies. Antibodies do not recognize the whole immunogen but only small regions (epitopes). Each type of antibody binds to its own inducing epitope. For example, lysozyme, an enzyme that degrades the carbohydrate coat of bacteria, induces several different antibodies, each of which binds to a particular epitope on the lysozyme molecule (Lodish et al., 2000). Although different epitopes on lysozyme differ greatly in their chemical properties, the interaction between lysozyme and antibody is complementary in all cases. In other words, the surface of the antibody’s antigenbinding site fits into that of the corresponding epitope as if they are molded together. The intimate contact between these two surfaces, stabilized by numerous noncovalent bonds, is responsible for the exquisite binding specificity shown by an antibody.

Epitopes
Immune cells do not interact with or recognize an entire immunogen molecule; instead, lymphocytes recognize discrete sites on the antigen called epitopes (antigenic determinants). Epitopes are the immunologically active regions of an immunogen which bind to specific membrane receptors for antigen on lymphocytes or to secreted antibodies. Interaction between lymphocytes and a complex antigen may involve several levels of antigen structure. In the case of protein antigens, the structure of an epitope may involve elements of the primary, secondary, tertiary, and even quaternary structure of the protein. In the case of polysaccharide antigens, extensive side-chain branching via glycosidic bonds affects the overall three-dimensional conformation of individual epitopes. Epitopes are small linear sequences of amino acid residues, branched sequences of carbohydrate, or “shape” sequences brought about by the folding of a protein molecule. The

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remaining part of the protein antigen molecule is the carrier for the epitope. An antigen such as bovine serum albumin has several different epitopes on its surface, each of which stimulates the cell having the appropriate receptor. Because an epitope usually comprises a sequence of approximately three to eight amino acid residues, antibodies should be regarded as site- or region-specific detection molecules instead of antigen-specific molecules.

ANTIBODIES
Immunohistochemical labeling with antibodies has become the most sensitive and powerful method for localizing antigens in situ and thus for characterizing cells and their components and their functions. In this context the importance of antibody specificity and selectivity for the antigen cannot be overemphasized. The specificity relies entirely on the properties of the primary antibody, independent of the procedure used for detection. Although both monoclonal and polyclonal primary antibodies can be generated or purchased, the former are preferred and are in much wider use because of their far greater specificity. The basic structure of an antibody molecule is Y-shaped, with the two tips designed to recognize and bind antigens (Fig. 2.1). The tips, through the disulfide bridge, are free to bend with respect to each other. This property increases the binding strength of the antibody for antigens that have multiple, adjacent antigenic determinants and for antigens that are closely packed together. The remainder of the antibody molecule enables it to interact with other proteins, preventing undesirable company. Antibodies are molecules secreted by terminally differentiated B cells (a type of lymphocyte) known as plasma cells. Nearly all rabbit primary antibodies and most mouse monoclonal antibodies are immunoglobulins (Igs). There are five classes of Igs that differ structurally and functionally. Immunoglobulin G (IgG) molecules are the major class of Igs in the blood, which are predominantly produced in the secondary immune response. Monoclonal antibodies have been termed magic bullets and hailed in publications as the cure for cancer. Belief in this idea was strengthened by the successful clinical results of mouse

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anti-idiotypic monoclonal antibodies in the treatment of lymphomas and leukemias (Levy and Miller, 1983) and by FDA approval in 1986 of the OKT3 anti-CD3 mouse monoclonal antibody for acute renal transplant rejection (Shield et al., 1996). However, this excessive optimism has been questioned because of adverse clinical and laboratory findings. For example, when rodent monoclonal antibodies were used therapeutically, a human antimurine antibody response developed in up to 50% of treated patients (Khazaeli et al., 1994). Effector functions of mouse antibodies also have proven less efficient in the human context (Gavilondo and Larrick, 2000). The biological half-life of these antibodies is shorter than that of human immunoglobulins. This characteristic of mouse antibodies limits their usefulness. These limitations can theoretically be overcome by using monoclonal antibodies of human origin (Thompson, 1988); however, human monoclonal antibodies from hybridomas and lymphocyte cell lines are very difficult to generate. Nevertheless, beginning in 1994, the FDA approved a number of antibodies to combat human diseases, including follicular non-Hodgkin’s B cell lymphoma, breast cancer, and rheumatic arthritis (Grillo-Lopez et al., 1999; Weiner, 1999; Maini et al., 1999).

Polyclonal Antibodies
Both polyclonal and monoclonal antibodies have advantages and limitations with regard to their generation, specificity, cost, and overall applications. Polyclonal antibodies possess higher affinity and wider reactivity but lower specificity. They have the advantage of detecting many types of epitopes and recognizing antigens of different orientations. Polyclonal antibodies show greater stability at varying pH levels and salt concentrations and are more useful for preadsorption controls. They are simpler to produce in a shorter duration, and there is no risk of loss of clones. In addition, large animals (e.g., rabbits and horses) can be used to recover large volumes of antibody-rich serum. However, a fresh batch of the serum is required when the original stock is exhausted. This replacement results in batch-to-batch variation, which may result in differences in antibody reactivity and titer (Nelson et al., 2000). Such differences result in a lack of reproducibility. Polyclonal antibodies are composed of multiple species of immunoglobulins directed toward several epitopes within a particular antigenic molecule. Moreover, only a minor proportion of the antibody present in the polyclonal antiserum is specific for the immunizing antigen. The remainder may consist either of antibodies produced by the animal in the past in response to previous antigenic stimuli or of antibodies against contaminating antigens present in the immunizing preparation (Mason et al., 1983). Even the antibodies in the polyclonal antiserum that are specific for the immunizing antigen are usually heterogeneous and are directed against a number of different epitopes on the immunizing antigen. However, although whole sera or whole IgG fractions of polyclonal antibodies often have problems, affinity purification against an antigen affinity column can dramatically improve the usefulness of the polyclonal reagents. Thus, a mixture of isoforms of antibodies to different epitopes is obtained. These epitopes are still relatively unique to the antigen involved. The main advantage is that 100% of the antibody in these preparations reacts with the antigen, often at multiple and therefore additive sites. This approach is analogous to mixing monoclonal antibodies to label different epitopes together. Although such reagents

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are not commonly commercially available, their usefulness in immunohistochemistry should not be minimized. The procedure to produce such reagents requires the ability to prepare significant amounts of the pure antigen, either a purified protein (or carbohydrate) or even a complex peptide. The antigen is covalently linked to the beads of a column (usually cyanogen bromide–activated Sepharose). The antibody preparation is passed over the column and the antibody reactive with the antigen sticks. The excess (usually 99% of the total IgG) (flow through) is then washed away from the column, and the specific antibody is eluted from the column selectively using acid, base, thiocyanate, or a high salt such as magnesium chloride. The eluted antibody is then neutralized and dialyzed to remove the salts. Such antibody usually represents only ~1% of the total IgG in the serum. This procedure is different from the so-called affinity-purified antibody in which protein A or protein G is used; the latter purifies IgG only and has no meaning in relation to selective antigen reactivity.

Production of Polyclonal Antiserum
The following procedure can be used to produce polyclonal antiserum in rabbits (Beltz and Burd, 1989). Preimmune blood is removed from the rabbit’s ear vein for later use in control experiments. The rabbit is immunized with of the antigen. If a carrier protein is used, the carrier and the antigen are injected together. One milliliter of the antigen (including the carrier) in buffer and 1 ml of Freund’s complete adjuvant are emulsified completely and injected subcutaneously into several locations on the rabbit’s back. Freund’s complete adjuvant contains ingredients that increase the rabbit’s immune response. The complete adjuvant includes saline, emulsifying agent, mineral oil, and killed mycobacteria. The mixture is injected once a week for 3 weeks, and then the animal is maintained for 3 weeks without additional injections. Approximately 25–40 ml of blood is removed from the animal’s ear vein to test for antibodies. One week after the first bleed the rabbit is boosted with half the antigen amount used earlier along with incomplete adjuvant; incomplete adjuvant does not contain the mycobacteria. Serum is again removed 2 weeks after this injection and tested for antibody response. The enzyme-linked immunosorbent assay is used to determine if the titer (or antibody concentration) of the serum is sufficiently high to establish antibody binding to the antigen. This 3-week cycle is repeated as long as necessary to obtain the antibodies desired. The peak response is generally achieved at the sixth to eighth injection (~5–7 months) after the initial immunization. If the rabbit is to be sacrificed, the final bleeding can yield 50–70 ml of serum. When the animal has made antibody in sufficient quantity, its serum can be used directly as the immunohistochemical reagent. It can be purified using an affinity column to remove nonimmunoglobulin proteins, extraneous antibodies, or to select antibodies that recognize a specific antigen. The use of affinity purified antibodies reduces nonspecific, background staining.

Affinity Chromatography
Antibody affinity chromatography is employed to isolate antigen-specific antibodies. The most common affinity matrix for coupling of molecules is cyanogen bromide–activated

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Sepharose. The following procedure can be used to purify antibodies raised against a particular protein (Javois, 1999).
1. Sprinkle 1 g of cyanogen bromide–activated Sepharose 4B (Pharmacia-LKB, Piscataway, NJ) over 20 ml of 10 mM HC1. The gel swells immediately. One gram of dry gel yields ~3.5 ml of hydrated matrix. 2. Wash the gel on a 50-ml coarse, sintered glass funnel four times with 50 ml of 10 mM HCl by repeatedly suspending the matrix in the HCl solution, and then drain using vacuum suction. These washing steps remove the additives present in the dry matrix. 3. Suspend 5 mg of rat immunoglobulin in 5 ml of coupling buffer A (100 mM 500 mM NaCl, and 200 mM glycine, pH 8.0). 4. Add this supension to the gel and mix by inversion overnight at 4°C in a capped 15-ml polycarbonate tube. Avoid mechanically stirring the gel to avoid damaging the gel matrix. 5. Pour the matrix into a sintered glass funnel and drain the gel; save the eluate to estimate the amount of antibody couple. Estimate the amount of protein bound to the column by subtracting the quantity of IgG that is eluted. The eluate should not contain more than 20% of the applied protein concentration. 6. Wash the matrix with 100 ml of coupling buffer A to remove any unbound ligand. 7. Suspend the matrix overnight at 4°C in 45 ml of 200 mM glycine (pH 8.0) and mix by inversion in a 50-ml capped tube to block any unreacted groups. 8. Drain and wash the gel with three cycles of alternating pH. First, suspend the drained gel in 50 ml of 100 mM sodium acetate (pH 4.0) and 500 mM of NaCl. Drain with vacuum suction and wash with 50 ml of coupling buffer A. Drain and repeat the alternating pH washes twice. 9. Suspend the gel in 20 ml of BBS buffer (dissolve 247.3 g of boric acid, 187 g of NaCl, and 75 ml of 10 M NaOH in 4 liters of distilled water, pH 8.0). The matrix is then ready for use in column chromatography. 10. Pack the matrix in a Poly Prep column (Bio-Rad), which can be stored at 4°C; it should not be allowed to warm up or dry out. 11. Attach the column outlet to a peristaltic pump, and wash the column with 5 ml of BBS at 0.5 ml/min. 12. Drain most of the BBS, leaving ~0.5 ml on top of the gel bed. 13. Apply 15 ml of rabbit antirat IgG to the column, and circulate the solution through the matrix at 0.2 ml/min for 3 hr at 4°C. 14. Drain the column as in step 12 and save the eluate, which may still contain some of the desired antibodies. The titer of the eluate can be tested for reapplication to the gel at the end of the first purification, although this may not be necessary. 15. Wash the matrix with ~10 column volumes of BBS until the absorbance of the eluate is <0.02 at 280 nm compared to the column buffer. 16. Remove the bound antibody with 5 ml of 100 mM glycine (pH 3.0) at 0.5 ml/min. of 1 M Tris-HCl buffer (pH 8.0). 17. Collect 1-ml fractions into tubes containing 18. Pool the IgG-containing samples and concentrate as necessary. The samples containing the highest absorbance at 280 nm should be pooled. Any precipitated antibodies can be removed by centrifugation at 10,000 g for 30 min at 4°C. The IgGs

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can be concentrated and stored at 4°C for weeks or at – 80°C for months and years. Avoid repeated freezing and thawing, which may denature the proteins.

Monoclonal Antibodies
Unlike polyclonal antibodies, monoclonal antibodies are directed against single epitopes consisting of very short sequences of amino acids. These antibodies are able to provide invaluable information about the molecular conformation of a particular epitope within a given antigen. However, even though a monoclonal antibody can recognize multiple molecules, its specificity remains intact. This phenomenon is due to the presence of very similar epitopes in different peptides and proteins. This subject is discussed in detail later in this chapter. The development of monoclonal antibodies has led to a new era of enhanced diagnostic and therapeutic modalities. The impact of this development on diagnostic imaging technologies has been dramatic, resulting in a revolution in modern diagnostic medicine. Recent advances in recombinant antigen preparation have further advanced the usefulness of these methods; almost any protein can be engineered to be expressed in nonnatural host cells. If monoclonal antibodies to a specific region or a specific epitope of an antigen are desired and the amino acid sequence of this region is known, synthetic peptides can be prepared for animal injection. In fact, synthetic peptides are currently being used for the injection of animals to generate antibodies for a specific region or a specific region epitope of an antigen. As a result, monoclonal antibodies of high affinity and specificity are being produced at a fast pace. These antibodies are being effectively employed for the detection and analysis of antigens, and are playing a key role in clinical diagnostic medicine. These developments are partially responsible for the exponential growth in our understanding of the physiology of human diseases. Monoclonal antibodies are produced only when necessary because their generation is difficult, time-consuming, and frustrating. Nevertheless, the most important reason for preferring them is their exquisite specificity, as ideally they recognize only one type of epitope. Moreover, these antibodies show a high biological half-life in blood and other tissues, rendering them effective for prophylactic use. The toxicity of infused monoclonal antibodies is expected to be low because of their biological nature. Thus, these antibodies are being used extensively and successfully in routine pathology laboratories to aid in the clinical diagnosis and treatment of malignant diseases. To produce monoclonal antibodies, lower doses of antigens are required for immunoresponse. The continuous culture of B cell hybridomas yields a reproducible and potentially inexhaustible supply of the monoclonal antibody; all batches are homogenous. Consequently, these antibodies allow the development of standardized procedures for clinical diagnosis. In addition, monoclonal antibodies are ideal for a complex mixture of antigens (e.g., membrane antigens), for scarce antigens, or when attempting to detect unique epitopes where purification is difficult or impossible (Beltz and Burd, 1989). Monoclonal antibodies do not have high affinities and so generally must be used at lower dilutions. Moreover, monoclonal antibodies are more expensive to generate or purchase than polyclonal antibodies. Exceptions to some of the advantages and limitations of polyclonal and monoclonal antibodies listed above are not uncommon. A large number of monoclonal antibodies are commercially available (see page 50).

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Specificity of Monoclonal Antibodies

Affinity is an important feature of monoclonal antibodies and is especially important for those antibodies used in clinical diagnosis. The measurement of antibody affinity provides an indication as to the strength with which a monoclonal antibody specifically binds to its target molecule. Another important characteristic of a monoclonal antibody is the specific location or region on the antigen to which the antibody binds. Although most monoclonal antibodies exhibit strong affinity and high specificity for epitopes, this is not true for all. In this context, the mechanisms responsible for reaction of antibodies with antigens in the tissues that have been processed for immunohistochemistry are exceedingly complex. The processing conditions under which an antigen is exposed to an antibody are critical in determining the specificity of an antibody. The processing conditions employed determine the reactive epitopes. Under certain conditions the epitope recognized may be other than that under study. Various aspects of antibody–antigen interaction, including the role of fixation, are discussed below. Monoclonal antibodies are directed against epitopes consisting of a small number of amino acids, which can be part of several types of proteins and peptides. Therefore, it is not uncommon for some monoclonal antibodies to label unrelated antigens in different tissues. Several examples are cited below. A monoclonal antibody against a monocyte-macrophage protein also binds to enamel proteins (Nakamura et al., 1991). Several monoclonal and polyclonal antibodies against osteocalcin (a bone protein) also cross-react with epitopes on cultured skin fibroblasts (Bradbeer et al., 1994). An anti–human proinsulin antibody crossreacts with both insulin and glucagon-secreting cells (Bendayan, 1995). Another example is the monoclonal antibody (C219) against the multidrug transporter (P-glycoprotein), which also reacts with the slow-twitch skeletal muscle myosin (Thiebaut et al., 1989). Because most of the immunohistological studies, including those cited above, are carried out using chemically fixed tissues, it is difficult to be certain whether the immunoreactivity is owing to shared epitopes (cross-reactivity) or epitopes resulting from protein crosslinking during fixation with an aldehyde. A recent study has demonstrated that antivimentin antibody (V9-S) cross-reacts with amelogenins in the glutaraldehyde-fixed rat hemimandibles (Josephsen et al., 1999). This immunoreaction is thought to be directed against the epitope generated by crosslinking of enamel proteins during fixation because this antibody binding occurs only after fixation. Moreover, any significant homology between vimentin and amelogenin is absent. Another example is PC10 antibody, which reacts with PCNA only after fixation (Willingham, 1999). Such reactions have been called nonspecific but selective. However, as long as these reactions are useful, they are both specific and selective. Considering the possibility of a monoclonal antibody reacting with more than one type of antigen or epitope, the positive staining should be interpreted carefully. Nonspecificity in the immunostaining can render correct interpretation difficult. This problem can arise due to nonspecific binding of antibodies and reagents, cross-reacting endogenous antibodies, and the presence of the same or similar epitopes in different antigens. In other words, when monoclonal antibodies show unexpected cross-reactivity, it is not clear whether this phenomenon is due to the presence of the same molecule in two different cell populations or to the presence of identical (or very similar) epitopes on quite different molecules. These difficulties are encountered in both frozen and paraffin sections. It should be

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noted, however, that the major effect of aldehyde fixation on the reaction of the antibody with the antigen is the inaccessibility of the antigen to the antibody. In other words, the crosslinking of cytosol proteins introduced by aldehyde fixatives creates a barrier for the antibody molecule to penetrate the cell and react with the antigen (epitope). Monoclonal antibodies generated against epitopes react with the native conformation of the antigen, which may be preserved by fixation. These antibodies are useful for immunohistochemistry, provided antigens are accessible to antibodies. In contrast, some other monoclonal antibodies preferentially react with denatured antigens after treatment with denaturing agents such as sodium dodecyl sulfate (SDS). Such antibodies generated by immunizations with isolated peptides usually are not well suited for immunohistochemistry (Willingham, 1999). Based on these observations, it is likely that most monoclonal antibodies are reactive under certain appropriate conditions used to prepare and incubate the specimens of interest. There are still other antibodies that are reactive with both native and denatured conformations of proteins. It is thought that these antibodies recognize peptide sequences exposed when proteins are denatured. On the other hand, polyclonal antibodies do not require optimal processing condition because a polyclonal antibody recognizes multiple epitopes. These antibodies may recognize an epitope other than that under study.

MIB-1 Monoclonal Antibody
Because MIB-1 monoclonal antibody is used extensively to determine the cell proliferation index, its applications are discussed below. This antibody detects the nuclear antigen Ki-67 expressed in proliferating cells but not in resting cells. The antibody reacts with the nuclei of cells in (first gap), S (DNA synthesis), (second gap), and M (mitosis) phases, but not in the or quiescent phases. The use of MIB-1 antibody is one of the simplest and most reliable labeling techniques for assessing the rate of proliferation of a neoplastic cell population. Thus, the antibody can be used to assess the growth fraction (i.e., the number of cells in cell cycle) of normal, reactive, and neoplastic tissues. However, be aware that in spite of the usefulness of the MIB-1 antibody in assessing the rate of cell proliferation, the classification of cancers (e.g., breast cancer) by the size of the primary tumor and the presence and extent of lymph node metastases does not adequately explain differences in the clinical outcome of individual patients. Cell proliferation indices are commonly used, along with other diagnostic parameters, to estimate the risk of recurrence of a cancer for individual patients. Therefore, it is important to understand the relationship between various indices of proliferation such as MIB-1 labeling index and detection by either in situ hybridization or polymerase chain reaction. This approach will lead to quality assurance in diagnosis. Considerable evidence is available indicating a close correlation between the growth fraction of proliferating cells, as measured by other techniques, and the number of cells stained using MIB-1 antibody. Thus, MIB-1 labeling index can be used in conjunction with other histological features to determine the potentially aggressive behavior of a tumor. For example, MIB-1 labeling index of the growth fraction correlates significantly with the bromodeoxyuridine (BrdU) index of DNA synthesis in S phase, and both indices correlate well with other parameters of tumor aggressiveness (Goodson et al., 1998). In many cases,

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but not in all, a correlation also exists between the label index and patient survival. However, although these indices are related, clinical comparison is necessary to determine which is the better prognostic marker for human breast cancer and other human cancers. A brief comment on the usefulness of the BrdU method is relevant. Currently, immunohistochemical detection of exogenously injected 5-bromodeoxyuridine several hours prior to animal sacrifice is widely used to assess proliferation state in murine tissues. This nucleotideanalog probe is integrated into the DNA of replicating cells during S phase (Selden et al., 1993). The probe can be subsequently detected immunohistochemically in paraffin-embedded tissue sections. However, the proportion of anti-BrdU stained cells is variable depending on the duration and frequency of BrdU injections before sacrificing the animal. A number of examples given below support the prognostic value of MIB-1 antibody. Evaluation of proliferation index in malignant mesothelioma can be performed using the MIB-1 antibody (Comin et al., 2000). In this case a correlation is found between the labeling index and survival. Malignant mesothelioma is a rare, aggressive, and frequently lethal tumor, usually associated with asbestos exposure. It should be noted that numerous prognostic factors (e.g., age, tumor stage, asbestos exposure, performance status, histological subtype, tumor angiogenesis, and proliferation index) are correlated with survival. Another example is the high proliferative index found in the esophageal small cell carcinoma using MIB-1 antibody, indicating aggressive behavior (Lam et al., 2000). Also, Ki-67 antigen labeling with MIB-1 antibody is a reliable method for estimating the proliferative activity in uveal melanomas after proton beam irradiation (Chiquet et al., 2000). The Ki-67 score is significantly correlated with prognostic variables (mitotic index and histological largest tumor diameter) and with radiation effects after proton beam irradiation. Furthermore, a higher Ki-67 score is found in uveal melanomas with metastasis than in tumors without metastatic evolution. Uveal melanoma is the most common primary adult ocular malignancy. Their ability to metastasize is well recognized. It should be noted that these views have not been accepted universally. Another example is the labeling of Ki-67 with MIB-1 antibody in benign and malignant apocrine lesions of the breast, which facilitates differentiation between benign and malignant breast apocrine lesions (Moriya et al., 2000). Both the number of positive cases and the percentage of positive tumor cells are significantly higher in malignant cases than in benign apocrine cases. Thus, Ki-67 immunohistochemistry can be an auxiliary method for determining the possible biological behavior of lesions and for the diagnosis and prognosis of patients with apocrine lesions of the breast. MIB-1 immunostaining in conjunction with microwave antigen retrieval is a beneficial adjunctive test when the morphological features are suggestive but not diagnostic for vulvar condyloma acuminatum (Pirog et al., 2000). Histopathological confirmation of condyloma acuminatum implies human papillomavirus (HPV) infection, which is sexually transmitted and confers an increased risk for synchronous or subsequent HPV-associated wartlike lesions elsewhere in the female genital tract. However, histopathological diagnosis of condyloma acuminatum is often based on architectural features that are not specific for HPV infection. To avoid this problem, the application of MIB-1 antibody is useful in the differential diagnosis of benign exophytic vulvar lesions because HPV-associated lesions show

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increased cellular proliferation. Limiting the diagnosis of condyloma to only those lesions with koilocytotic atypia will result in underdiagnosis, and categorizing equivocal wartlike lesions of the vulva as c/w condyloma acuminatum is associated with substantial overdiagnosis. Complete concordance was found between MIB-1 positivity and detection of HPV by both in situ hybridization and polymerase chain reaction (Pirog et al., 2000); however, MIB-1 positivity is more sensitive than the other two techniques. Antibody MIB-1 also shows cross-reactivity. It has been demonstrated, for example, that this antibody shows strong staining of cell membrane and cytoplasm in hyalinizing trabecular adenoma of the thyroid gland (Hirokawa and Carney, 2000). In contrast, this antibody does not show similar immunoreactivity with papillary carcinoma. This information provides a morphological difference between hyalinizing trabecular adenoma and papillary carcinoma, which is important because it has been suggested that these two types of tissues are closely related tumors (Fonseca et al., 1997). This benign tumor does share histological features, such as intranuclear cytoplasmic invaginations, nuclear grooves, and psammoma body–like formations, with papillary carcinoma. Thus, MIB-1 antibody can be diagnostically useful in differentiating the benign tumor from papillary carcinoma. Note that MIB-1 antibody fails to react with Ki-67 antigen in the tissue fixed with formaldehyde or Kryofix in the absence of heat pretreatment. Because Kryofix is a nonprotein, crosslinking fixative, breakdown of protein crosslinkages is not responsible for the availability of Ki-67 antigen in this case. On the other hand, MIB-1 antibody readily reacts with Ki-67 antigen in tissue fixed with either of these two fixatives with heat pretreatment. It is apparent that in this case the effect of heat treatment is due to factors other than breakdown of protein crosslinkages. Although MIB-1 antibody is a reliable tool for determining proliferating cells in human tissues, it does not react with the homologous mouse antigen. Therefore, this antibody is useless in experimental pathology using mice as the model system. Because the use of murine tumor models has steadily increased, there is a growing need for a proliferation marker for routinely processed paraffin-embedded murine tissues. Such a marker is monoclonal antibody MIB-5, which is raised against bacterially expressed parts of the human Ki-67 cDNA (Schlüter et al., 1993; Gerlach et al., 1997). Recently, Birner et al. (2001) have shown that MIB-5 detects Ki-67 antigen in formalin-fixed, paraffin-embedded murine tissues, equivalent to MIB-1 staining of human tissues.

Production of Monoclonal Antibodies

Before presenting the procedure for generating monoclonal antibodies, it is relevant to define the terms hybridoma and monoclonal antibody. When an immune response is provoked by an immunogen, numerous antibodies are produced against different parts or regions of the immunogen. These are termed antigenic determinants, or epitopes, and they usually consist of six to eight amino acids. Most antibodies recognize and interact with a three-dimensional shape of an epitope composed of discontinuous residues brought together by the folding of a molecule (usually a protein antigen). Antibodies can also recognize linear stretches of amino acids or continuous residues (an epitope). An antibody of unique specificity derived from a single B cell is termed a monoclonal antibody.

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An immunogen induces antibodies from many B cell clones, producing a polyclonal antibody response. In contrast, the propagation of an isolated B cell clone produces an antibody of single specificity. However, the problem is that in tissue culture medium, B cells die within a few days of their isolation from, for example, a mouse spleen. To circumvent this problem, immortality can be conferred on B cells by means of viral transformation; Epstein-Barr virus can be used. Alternatively, fusion to cancerous cells is carried out to generate hybrids or hybridomas. Generally, the former procedure is used to immortalize peripheral blood B cells and produce human monoclonal antibodies, while myeloma cells are used to produce murine monoclonal antibodies. Although several recombinant procedures can be employed to produce monoclonal antibodies, the following protocol is used to generate murine monoclonal antibodies. It consists of four steps: immunization, fusion and selection, screening, and characterization (Nelson et al., 2000). 1. Immunization. Immunogen protein linked to a carrier protein (e.g., keyhole limpet hemocyanin) is used for the primary immunization of Balb/c mice. An immunogen is delivered in conjunction with Freund’s complete adjuvant. Adjuvants are used to enhance the stimulation of antibody production. They act by making the antigen either more particulate or more insoluble, thus holding the antigen in a depot and releasing it slowly over a long period of time. These substances may also stimulate the proliferation of B cell precursors in spleen, lymph nodes, and liver. Freund’s medium is a mixture of mineral oil and antigen that is emulsified in lanolin and to which killed tubercle bacilli have been added. The latter addition increases the efficiency of the adjuvant by stimulating the proliferation of macrophages. Freund’s adjuvant is unsuitable for human use. Two milliliters of the adjuvant is placed into a 13 × 100 mm test tube. A small quantity of antigen is added and emulsified with a 5-ml syringe and a 20-gauge needle. Small amounts of antigen continue to be added. Small amounts of antigen are added and emulsification is continued until a total of 2 ml of antigen has been added. The emulsion must be water-in-oil (Burrell and Lewis, 1987); it is satisfactory when a drop placed on a water surface does not spread. For rabbits, 2 ml of this preparation is injected intramuscularly in each hind leg or subcutaneously in each of two sites at the back of the neck. Animals start responding after 3 to 4 weeks. Regular boosting may be needed to augment polyclonal response, which is monitored using tail bleeds that provide sufficient serum to make sure the antibody titer to a desired antigen, using the enzyme-linked immunosorbent assay (ELISA) detailed below. Ten wells in a row of a plastic microtiter plate are each coated with of ovalbumin diluted coating buffer: Coating buffer
3.18g 5.84 g 1,000 ml 9.5

Distilled water Adjust pH to

Well 11 is skipped and well 12 is coated with of the antigen. Wells in the second row are filled with of coating buffer to serve as blanks. Incubation is carried out for 30 min at 37°C.

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While the incubation is continuing, an antiovalbumin is titrated beginning at 1/50 through 10 dilutions in 0.25-ml quantities. The serum is titrated in the diluting buffer with a micropipette. Diluting buffer
Tween 20 NaCl Distilled water Adjust pH to
0.5 ml 8.5 g 1,000 ml 7.2

To raise pH, add 30 ml of to lower pH, add 100 ml of this solution. All wells in a row are carefully rinsed with a wash buffer simultaneously using a well-washing device (Nunc Immunowash). Wash buffer is the same as diluting buffer but without Tween 20. The rinsing is done at least eight times, and all buffer after the last rinse is removed. Starting with the last serum dilution, is transferred to well 10 of each row. Then, of the next serum dilution is transferred to well 9 of each row, and so on until one has reached the starting dilution, of which is transferred to each well 1. Incubation is carried out for 30 min at 37°C. This is followed by thorough rinsing with the wash buffer using a well-washing device as indicated above. One hundred microliters of commercial goat antirabbit globulin conjugated to horseradish peroxidase (diluted 1/500 diluting buffer) is added to every well. Incubation is done for 30 min at 37°C, followed by washing, and of peroxidase substrate in freshly prepared substrate buffer is added to each well. Substrate buffer
Citric acid Distilled water Adjust pH to 11.85g 11.73 g 1,000 ml 4.0

Substrate
Ten milligrams of 2,2´-azino-di-(3-ethylbenzthiazoline sulfonic acid) diammonium salt is dissolved in 50 ml of substrate buffer containing of 30% hydrogen peroxide. Only freshly prepared substrate should be used. Incubation is carried out for 30 min at 37°C, followed by visual plate reading on a 1+ to 4+ basis or at if a microtiter plate reader is available. The optical density of the instrument is set to zero with the antigen control (well 12 in the first row). Any optical density in the antibody controls (bottom row) is subtracted from the corresponding test well above. The proteins are delivered subcutaneously. Regular boosting is needed to augment polyclonal response, which is monitored using tail bleeds that provide sufficient serum to make sure the antibody titer to a desired antigen, using the enzyme-linked immunosorbent assay (ELISA). Boosting also encourages immunoglobulin class switching and the generation of higher affinity antibodies through somatic hypermutation. Generally, IgG monoclonal antibodies are preferred because they are less prone to degradation and more useful

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as therapeutic reagents. Antigenically responding B cells are removed aseptically from the spleen or lymph node to obtain cells for hybridization. 2. Fusion and selection. The murine splenic B cells are fused with histocompatible myeloma cells such as Sp2/0. The myeloma cells are preselected for a deficiency in the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) by culturing in medium containing 8-azaguanine. The B cells are mixed with HGPRT-negative myeloma cells and a fusing agent such as polyethylene glycol. The mixing and centrifugation steps generate myeloma-splenic B cell hybridomas. These hybrid cells are plated into tissue culture wells. Unfused myeloma cells are removed using a selective medium containing hypoxanthine, aminopterin, and thymidine (HAT); all unfused myeloma cells will die. Hybridomas are carefully examined with an inverted microscope. Once established, the hybridoma colony will continue to grow in the culture medium (such as RPMI-1640 containing antibiotics and fetal bovine serum) and produce antibodies. Approximately 1 month post fusion, hybridomas can be propagated in HT medium (hypoxanthine and thymidine). 3. Screening. Primary screening is necessary to eliminate nonspecific hybridomas as soon as possible. Screening is also used to test the hybridoma culture supernatant for antibody reactivity and specificity. As an example, an Epstein-Barr virus associated protein is coated onto plastic ELISA plates. After incubation of hybridoma culture supernatant, secondary enzyme-labeled conjugate and chromogenic substrate, a colored product indicates a positive hybridoma. Alternatively, immunocytochemical screening can be used. It is preferable to test hybridomas when at least three-quarters of them are confluent. 4. Characterization. The reactivity, specificity, and cross-reactivity of the potential monoclonal antibody can be analyzed by using culture supernatant or a purified immunoglobulin preparation. It may be necessary to redone hybridomas by limiting dilution because the original colony might contain at least two populations of fused B cells. In the absence of such an analysis, the presence of antibodies of different class, specificity, and affinity might yield ambiguous results. Characterization also provides the opportunity to test against a wide panel of related antigens or tissue preparations. This is important especially for histopathological studies. Once the purification of the hybridoma is established, bulk production of a monoclonal antibody can be obtained using surface-expanded tissue culture flasks. It should be noted, however, that although a hybridoma may be the fused product of a single B cell and produce a monoclonal antibody of refined specificity, such an antibody in some cases can cross-react with other antigens or exhibit dual or multiple specificity. The phenomenon of cross-reactivity is discussed on page 48. It is relevant to explain the use of the letters CD as a prefix to monoclonal antibodies. Lymphocytes possess many different surface proteins, each of which possesses many distinct epitopes. To classify these lymphocyte antigens, a numbering system has been established that clusters molecules having similar epitopes. Thus, all monoclonal antibodies that detect the epitopes on a single antigen are assigned to a numbered cluster of differentiation (CD). In most cases a defined CD denotes a protein of specific function. For example, the protein called CD4 is associated with cells (lymphocytes) that help the immune response, while CD8 is found on cells that suppress the immune response.

Bivalent and Bispecific Monoclonal Antibodies in Cancer Therapy
Bivalent and bispecific monoclonal antibodies have many practical applications, including immunodiagnosis and immunotherapy. Bivalency can allow antibodies to bind

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to multimeric antigen with strong avidity, and bispecificity facilitates the crosslinking of two antigens, for example, in recruiting cytotoxic T cells to mediate killing of a tumor cell. Bivalent (IgG) antibodies can be derived from hybridomas, and bispecific antibodies by fusion of two hybridomas with two different specificities. Bispecific antibodies can form useful immunotherapeutic tools in cancer treatment. The limited space available in this volume does not allow detailed discussion of the applications of these antibodies. Important in vivo animal studies, which administer bispecific antibodies, and clinical trials using these antibodies are listed by Koelemij et al. (1999). Bispecific antibodies have been known for a long time. The first antibody with dual specificity was described approximately 40 years ago (Nisonoff and Rivers, 1961). The potential usefulness of these antibodies in the treatment of malignancies becomes clear when one considers that some therapeutic applications of monoclonal antibodies in patients with cancer have shown disappointing results. However, in general, monoclonal antibodies are thought to achieve antitumor effects by inducing antibody-dependent cellular toxicity or complement-mediated cytotoxicity. Considerable experience has been gained in using monoclonal antibodies in patients with cancer, especially in the treatment of hematological malignancies (Matthews et al., 1995). Nevertheless, the application of many monoclonal antibodies does not completely eliminate tumor cells. The cell surface expression of complement-deactivating molecules is thought to be the escape mechanism used by tumor cells in this incomplete elimination. Because many potentially useful monoclonal antibodies do not possess the appropriate isotype and so are unable to activate human complement and/or trigger on human cells, treatment strategies are needed. One such strategy is the application of bispecific monoclonal antibodies that exploit the specificity of monoclonal antibody and ensure activation of cellular cytotoxic mechanisms (Fanger et al., 1993). Bispecific monoclonal antibodies are artificially developed antibodies with antigenbinding sites physically linked to different specificities. It is thought that bispecific monoclonal antibodies activate the cellular immune response by crosslinking immune cells to tumor cells, thus circumventing the proper structures for tumor cell–immune cell interactions (Koelemij et al., 1999). These antibodies are effective in low concentrations in vivo. For example, Kufer et al. (1996) have combined the anti-CD3 specificity directed against T cells in a bispecific monoclonal antibody, with the specificity against the tumor-associated 17-1A antigen. This antibody could be a major improvement, for example, in the therapy for disseminated micrometastatic tumor cells. Bispecific monoclonal antibodies are being evaluated in phase I and II studies in a variety of malignant diseases in the fields of hematooncology and solid tumors. It is likely that in the next decade immunotherapy using bispecific monoclonal antibodies will have a place, complementary to the current modalities such as surgery, chemotherapy, hormone therapy, and radiation, in the treatment of malignancies.
Development of Bispecific Antibodies

The earliest bispecific antibodies were obtained by dimerization after mild oxidation of a mixture of Fab fragments of polyclonal antibodies of two different specificities. Bispecific antibodies were also obtained by crosslinking two monoclonal antibodies using succinimidyl-3-2-pyridyldithiol-propionate, resulting in heteroaggregates of two monoclonal antibodies (Staerz et al., 1985). Although this is an easy method with a high yield,

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the products are heterogeneous, ill defined, and large. Hybrid antibodies were also produced by chemical processing of Fab fragments to reconstitute (hybrid) dimers (Brennan et al., 1985). These antibodies are free of mono specific contaminants and thus avoid many unwanted side reactions. The most commonly used technique to produce bispecific antibodies from two monoclonal antibodies is by fusing two hybridoma cell lines by conventional cell fusion procedure (Staerz and Bevan, 1986). These cells produce all possible combinations of the heavy and light chains of both antibodies, including the desired bispecific antibody. A limitation is that only part of the antibodies is the desired bispecific monoclonal antibody; therefore, further purification is necessary (Van Ravenswaay et al., 1993). Currently, mostly molecular biological techniques are used to obtain bispecific monoclonal antibodies. By focusing on a genetic approach, molecules having the appropriate binding regions as well as bispecificity can be prepared. For example, a so-called leucine zipper technique has been employed for producing bispecific monoclonal antibodies (Kostelny et al, 1992). Leucine zipper peptides of the transcription factors Fos and Jun preferentially form heterodimers. In a genetic construct the sequences of the tail of one monoclonal antibody are replaced by the sequences from the leucine zipper region of Fos, and the sequences of the other monoclonal antibody are replaced by the sequences of the Jun leucine zipper. The murine myoloma cell line Sp2/0 is transfected with these constructs, resulting in the production of heterodimers of Other methods have focused on obtaining single-chain bispecific molecules. These molecules consist of two variable domains connected by a polypeptide spacer of two monoclonal antibodies, coupled by a linker (Traunecker et al., 1992). Another approach to construct small bivalent antibody fragments is through the dimeric antibody fragments (diabodies) (Holliger et al., 1993). The diabodies can be easily obtained from bacteria, can be expressed in high yield, and lack the Fc portions of the whole immunoglobulin. These antibody fragments with two antigen-binding sites comprise a heavy-chain variable domain connected to a light-chain variable domain on the same polypeptide chain. Using a linker that is too short to allow pairing between the two domains on the same chain forces the domains to pair with the complementary domains of another chain and create two antigen binding sites. It should be noted that antibody fragments are often preferable to complete antibodies, as the Fc region of antibodies can lead to undesirable targeting to cells expressing Fc receptors.

RECOMBINANT ANTIBODIES
A more recent strategy for cancer diagnosis and therapy is the application of genetically engineered antibodies. This approach is logical because antibodies are the paradigm for the design of high-affinity protein-based binding reagents. This technology includes chimeric and humanized antibodies, antibody libraries, and transgenic organisms as bioreactors (Gavilondo and Larrick, 2000). During the last 10–15 years, important advances have been made in the design, selection, and production of new types of recombinant antibodies. Recombinant antibodies have been reduced in size, dissected into minimal binding fragments, and rebuilt into multivalent high-avidity reagents (Hudson, 1999). Recombinant antibody fragments have also been fused to radioisotopes and with a variety

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of molecules, including enzymes for prodrug therapy, toxins for cancer treatment, viruses for gene therapy, cationic tails for DNA delivery, liposomes for improved drug delivery, and biosensor surfaces for cancer diagnosis and therapy for real-time detection of target molecules. For clinical diagnostic applications, antibody fragments alone (without Fc) can provide the full range of in vitro immunoassays through to in vivo tumor-targeting reagents. In fact, recombinant antibodies and their fragments have entered the clinic, both for cancer diagnosis and for therapy. These reagents represent more than 30% of all biological proteins undergoing clinical trials for diagnosis and therapy. Clinical results confirm that these new antibodies, directed at the appropriate tumor markers (e.g., CD20 and HER-2), can control diseases without apparent side effects (Cragg et al., 1999). Innovative selection methods have enabled the isolation of high-affinity cancer-targeting and antiviral antibodies, the latter capable of redirecting viruses for gene therapy applications (Hudson, 1999). It is now possible to select high-affinity antibody fragments directly from a viral culture rather than from a live mouse. One significant advantage of this new technology is the isolation of antibodies with new binding specificities against hitherto refractory antigens, thus avoiding the limitations inherent in the mammalian immune response (De Haard et al., 1999). In addition, bispecific antibodies and related fusion proteins have been produced for cancer immunotherapy, effectively enhancing the human immune response in anticancer vaccines and T cell recruitment strategies. More recently, a novel technique has been developed for high-throughput screening of recombinant antibodies, based on the creation of antibody arrays (De Wildt et al., 2000). This method uses robotic picking and high-density gridding of bacteria containing antibody genes followed by filter-based ELISA screening to identify clones that express binding antibody fragments. This approach can screen thousands of different antibody clones at a time against a large number of different antigens. Thus, antibodies against impure proteins and complex antigens can be isolated. However, because a cellular extract contains thousands of different proteins, the detection sensitivity of this filter-screening technique requires considerable improvement to be useful for fingerprinting differentially expressed proteins. The Food and Drug Administration has approved the use of engineered therapeutic antibodies. This technology is expected to be an important instrument in the toolbox of the molecular biologist. Although it is not exactly clear how monoclonal antibodies damage tumors in vivo, it is thought that the ability of the antibodies to crosslink membrane receptors and generate intracellular signals is part of the mechanism controlling the tumor growth.

ANTICANCER MONOCLONAL ANTIBODIES
Interest in the use of monoclonal antibodies in diagnosing and treating cancer has undergone a resurgence during the last decade. The most important reasons for the renewed interest are (Murray, 2000): (1) the development of human or humanized antibodies through recombinant techniques, resulting in the decrease or elimination of immunogenicity, (2) approval of monoclonal antibodies (trastuzumab and rituximab) for

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cancer treatment by the Food and Drug Administration, and (3) antitumor effects of monoclonal antibodies, especially on solid tumors as shown by clinical trials when used alone or in combination with chemotherapy or radiotherapy. Trastuzumab (Herceptin; Genentech, South San Francisco, CA) is a humanized monoclonal antibody that recognizes the human oncoprotein HER-2/neu, which is overexpressed in some breast cancers and other tumors. Rituximab (Rituxan; Genentech, South San Francisco, CA, and I DEC Pharmaceuticals, San Diego, CA) is a genetically engineered chimeric monoclonal antibody containing murine light- and heavy-chain and kappa light-chain constant regions. It binds to CD20, a differentiation antigen found exclusively on B cells and on more than 95% of B-cell non-Hodgkin’s lymphoma, but not on hematopoietic stem cells, preB cells, normal plasma cells, or other tissues. Monoclonal antibodies can mediate tumor destruction by both direct and indirect mechanisms. Direct mechanisms include (1) increased induction of apoptosis resulting from binding to calcium channels and (2) inhibition of ligand binding and suppression of transcription factors within the tumor cells resulting from binding to growth factor receptors (e.g., EGFR and HER-2). Monoclonal antibodies can also destroy tumor cells indirectly through immunological mechanisms such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity (Murray, 2000). The type of antigen to which the antibody binds is also relevant. The most effective monoclonal antibodies are those that bind to specific receptors and possess the strongest biochemical and/or immunological effects.

ANTIBODY CROSS-REACTIVITY
The ability of antibodies to bind molecules other than those molecules used as the immunogens is well known. Such a binding is termed cross-reactivity. Cross-reactivity is sometimes a source of confusion in the interpretation of immunohistochemically stained preparations because it cannot be detected by the usual controls for specificity. Although the antigen-antibody reaction is highly specific, in some cases even a monoclonal antibody elicited by one type of antigen can cross-react with another type of antigen. In other words, two closely similar proteins may react with an antibody raised by only one of them. There are a number of reasons for cross-reactivity. This problem arises when an antibody-combining site recognizes more than one antigenic determinant due to similarity in shape of different antigens. The cross-reaction can occur when the antigenic determinant site is a sequence of amino acids common to more than one antigen. This commonality can occur when the antigenic determinant is conserved in a family of proteins. Many proteins possess homologies in their amino acid sequences and thus show immunological cross-reactivity. Cross-reaction can also occur when two different antigens share an identical epitope or if antibodies specific for one type of epitope also bind to an unrelated epitope possessing similar chemical composition. However, the antibody-binding affinity for the crossreacting epitope is usually less than that for the original epitope. Processing conditions can expose an epitope related to the epitope under study but less so the former epitope. Careful evaluation of a given monoclonal antibody and its determinant can be accomplished by epitope mapping (Nelson et al., 1997).

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Another reason for cross-reactivity is the use of less than pure protein or conjugated or fusion proteins as immunogens; such immunogens produce a heterogenous population of the antibody having considerable cross-reactivity to the contaminants (Javois, 1999). Cross-reactivities to the carrier protein to which the antigen has been conjugated or fused can be removed by affinity chromatography; however, a possible disadvantage of this method is that the most desirable immunoglobulins having the highest affinity bind the tightest and are difficult to recover. These problems can be prevented and increased antibody specificity can be achieved by using synthetic peptides or protein fragments as eliciting antigens. Another potential source of cross-reactivity is the presence of Fc receptors in cells or tissues, which bind the Fc region of the primary or secondary antibodies. These nonspecific sites can be blocked with normal serum or nonimmune immunoglobulins. When a secondary antibody is used for detection, the normal serum or immunoglobulin for blocking should be from the same species as the secondary antibody. Undesirable or nonspecific staining can also be the result of impure reagents used in the staining. This background staining can be prevented by using purified reagents and optimizing conditions for tissue processing including staining. Nonspecific binding can also occur owing to ionic interactions with other proteins or organelles in the tissue (Grube, 1980). These interactions can be minimized by diluting the antibody and by increasing the salt concentrations in the diluent and the rinsing solutions (Javois, 1999).

POLYREACTIVE ANTIBODIES
Polyreactive antibodies are naturally occurring antibodies. They are primarily from IgM but also from IgG and IgA isotypes. Polyreactive antibodies are capable of reacting with a variety of antigens that may differ among themselves. Unlike classic autoantibodies, which react with specific host antigens, polyreactive antibodies react with specific host antigens. Polyreactive antibodies react with endogenous as well as exogenous antigens. The production of polyreactive antibodies is independent of antigen immunization. The affinity of these antibodies for different antigens varies but is relatively low compared with the affinity of monoclonal antibodies elicited by a mature antigen-driven response (Chen et al., 1995). It is thought that polyreactive antibodies have physiological relevance. These antibodies probably are involved in defense distinct from their counterpart monoreactive antibodies. Polyreactive antibodies constitute a first line of defense against invading microorganisms by enhancing phagocytosis or complement-mediated lysis or by amplifying an ongoing specific antibody response. In other words, polyreactive antibody–producing/antigen binding cells might play a role in the development and maintenance of immunological tolerance. It is also possible that polyreactive antibodies play a role in the homeostasis of all internal biological systems. These antibodies are known to bind to antigens in the blood and rapidly clear from the circulation (Sigounas et al., 1994). Various assays, including ELISA, immunoblots, and chamber ELIspot, have been employed for demonstrating polyreactivity of preimmune antibodies in both mice and humans (Ternynck and Avrameas, 1986; Quan et al., 1997; Klimman, 1994). In a complex biological matrix, polyreactive antibodies are bound by components of the matrix, leaving

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only a small fraction of the antibody free in solution. This antibody is unmasked during affinity purification and becomes detectable by ELISA. It is possible that limited denaturation of the antibodies occurs during purification, altering their monospecificity. For example, it has been demonstrated that the binding site of M11, a murine, monoclonal antibody, is altered after purification at a low pH (2.2), resulting in its binding to many different proteins (McMahon and O’Kennedy, 2000). The inference is that such a polyreactivity represents an acquired, artificial characteristic.

COMMERCIAL SOURCES OF ANTIBODIES
Antibodies are available from the following sources: Affinity Research Products Ltd. (Exeter, U.K.) AMAC, Inc. (Westbrook, ME, U.S.A.) Becton-Dickinson (Mountain View, CA, U.S.A.) Biochemicals AG (Augst, Switzerland) Biogenesis, Inc. (Sandown, NH, U.S.A.) Biogenics (San Ramon, CA, U.S.A.) Biomedical Technologies (Stoughton, MA, U.S.A.) Bioproducts (Indianapolis, IN, U.S.A.) Biotrend Chemikalien GmbH (Cologne, Germany) Biozol Diagnostica GmbH (Eching, Germany) Boehringer Mannheim (Indianapolis, IN, U.S.A.) Cambridge Biosciences (Cambridge, U.K.) Calbiochem/Oncogene Research Products (Cambridge, MA, U.S.A.) Chemicon (El Segundo, CA, U.S.A.) Cis Bio International (Sur Yvette, France) Coulter Clone (Miami, FL, U.S.A.) Dako (Carpinteria, CA, U.S.A.; Glostrup, Denmark) Dianova (Hamburg, Germany) EuroDiagnostica (Amersfoort, The Netherlands) Fitzgerald (Concord, MA, U.S.A.) HistoCIS (Marseilles, France) Immunonuclear Corp. (Stillwater, MN, U.S.A.) Immunotech S.A. (Marseilles, France) Incstar (Stillwater, MN, U.S.A.) Labsystems (Chicago, IL, U.S.A.) LabVision/NeoMarkers (Fremont, CA, U.S.A.) Lipshaw (Pittsburgh, PA, U.S.A.) Loxo (Dossenheim, Germany) Monosan (Uden, The Netherlands) Neomarkers (Fremont, CA, U.S.A.) Nichirei Ltd. (Tokyo, Japan) Novocastra (Newcastle upon Tyne, U.K.) Ortho-Clinical Diagnostics (Amersham, U.K.)

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Pharmacia, Biotech (Uppsala, Sweden) Pharmigen (San Diego, CA, U.S.A.) Polysciences (Warrington, PA, U.S.A.) Progen (Heidelberg, Germany) QED Bioscience Inc. (San Diego, CA, U.S.A.) R & D Systems (Abingdon, U.K.) Rockland Inc. (Gilbertsville, PA, U.S.A.) Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.) Serotec (Oxford, U.K.) Transduction Laboratories (Lexington, KY, U.S.A.) Ultraclone (Isle of Wight, U.K.) Vector Laboratories (Burlingame, CA, U.S.A.) Wak-Chemie (Bad Homburg, Germany) Wako Chemicals GmbH (Neuss, Germany) Zymed Laboratories (San Francisco, CA, U.S.A.)

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Chapter 3

Fixation and Embedding

Because chemical fixation prevents cellular disintegration, it is the foundation for almost all microscopic studies. It is the most widely used method for preserving specimens for light and electron microscopy. The reasons for its universal use are that it adequately preserves many cellular components, including soluble and structural proteins; prevents autolysis and displacement of cell constituents, including antigens and enzymes; stabilizes cellular materials against deleterious effects of subsequent preparatory treatments, including incubations; and facilitates conventional staining and immunostaining. Fixation also allows the clarity of structural details shown by photomicrographs and electron micrographs, and it can be applied easily. No other tissue preservation method can claim these advantages. Freezing is a useful adjunct to chemical fixation. The biochemical reactions of formaldehyde and glutaraldehyde with proteins are discussed here and in more detail elsewhere (Hayat, 1986, 2000a). An understanding of the effects of fixation on antigens and cell morphology is a prerequisite to accepting the validity of immunohistochemistry. It is necessary to know the extent of antigen preservation or destruction due to fixation and dehydration-embedding. The preservation of antigenicity is adversely affected by any type of chemical fixation because epitopes may be masked sterically by the surrounding soluble proteins in the tissue fluid. The fixative may crosslink another molecule (usually a protein) directly to the epitope or in the vicinity of the antigen. The former may allosterically alter the epitope configuration, inhibiting its recognition by the antibody. The latter may block the accessibility of the antibody to the antigen. In addition, the antigen may be degenerated by direct crosslinking with the aldehyde groups of the fixative. All of these changes may be reversible or irreversible or partly reversible or irreversible.

FORMALDEHYDE
Formaldehyde, a monoaldehyde, is preferred over glutaraldehyde (a dialdehyde) because the latter introduces mostly irreversible protein crosslinks, masking the epitopes. Formaldehyde penetrates rapidly into the tissue but crosslinks proteins slowly. Most of these crosslinks are reversible, and therefore masked epitopes can be easily unmasked by treatments such as heating. However, for better preservation of cell morphology, a mixture of formaldehyde (4%) and glutaraldehyde (0.01–0.1%) can be tried. Bouin’s fixative with its acidic pH should not be used.
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Reversibility of protein crosslinkages introduced by formaldehyde has been demonstrated in different biological systems. For example, formaldehyde has been applied at low concentrations as a reversible crosslinker for nearest neighbor studies of histone-histone and DNA-histone complexes (Russo et al., 1981). It has also been shown that fixation of polyheads of the bacteriophage with 5% formaldehyde can be reversed up to 86% with acetic acid and sodium borohydride treatment (Baschong et al., 1983). This study was carried out for stabilizing labile structures during both isolation and purification, followed by reverse fixation by acidification so that the protein constituents of these structures can be characterized by gel electrophoresis. However, acid treatment may not allow the recovery of the original protein structure in its native form of assembly.

Nature of Formaldehyde Solution
Aqueous formaldehyde as used for fixation contains mostly methylene glycol (~99%), its oligomers, and small amounts of formaldehyde. The proportion of the oligomers present depends inversely on the temperature. Formaldehyde solution cannot be obtained without the formation of methylene glycol. It is not the formaldehyde molecule that is primarily responsible for rapid penetration into the tissue but methylene glycol, which is the major component of formaldehyde solution. At concentrations of 2% or less, the formaldehyde in solution is present practically only as the hydrated monomer Methylene glycol is formed by the reaction between formaldehyde and water:

To maintain chemical equilibrium, more formaldehyde is found through the dehydration reaction:

Elevated temperatures favor the dissociation of methylene glycol to formaldehyde during fixation. In addition to the small amount of formaldehyde originally present in the aqueous solution, a little more is formed from the methylene glycol. However, formaldehyde component reacts very slowly with cellular proteins, and then it is slowly exhausted. This means that the interior of the tissue block after fixation for 4–6 hr at room temperature is exposed mainly to methylene glycol; therefore, this portion of the tissue is fixed by ethanol during dehydration, resulting in the coagulation of proteins. Rapid and uniform fixation throughout the tissue block with formaldehyde can be obtained at high temperatures, for example, in a microwave oven. Such temperatures enhance the speed and extent of formaldehyde reaction with proteins by dissociating the methylene glycol to formaldehyde as well as by depolymerizing the oligomers of methylene glycol (Boon et al., 1988).

Mechanism of Fixation with Formaldehyde
Like glutaraldehyde, formaldehyde is an additive fixative used for preserving tissues for light microscopy (Hayat, 2000a). When formaldehyde reacts with an amino acid that

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has an available hydrogen site, a reactive hydroxymethyl group is produced, which is the addition product. Subsequently, a second hydrogen site may react with this addition product, yielding a methylene bridge between two amino acids in the protein. As a result of this interaction a large number of hydroxymethyl groups are produced, utilizing reactive species on the side chains of amino acids or directly from the peptide bond (Table 3.1). In other words, additional reactions of formaldehyde with the hydroxymethyl groups and previously unreacted amino acid side chains form the methylene bridges that are the protein crosslinks of formaldehyde fixation. Formaldehyde reacts through several steps to form a methylene bridge between two neighboring amino groups of amino acids. Neutral pH favors the formation of such bridges. The primary reason for using neutral-buffered formalin is that at this pH hydrogen sites in peptide molecules are available for linkage because they are in an uncharged state. In contrast, an acid pH induces formation of charged amino groups that lack reactive hydrogen sites. Nonscientific reasons for using formalin are that it is inexpensive, and easily available, and diagnostic pathologists and technicians have been trained to use it. Formaldehyde introduces both intramolecular and intermolecular crosslinks between proteins involving hydroxymethylene bridges, which change the three-dimensional structure of proteins. Such changes involve the tertiary and quaternary structures of proteins, whereas the primary and secondary structures are little affected. It has been shown that the secondary structure of purified protein molecules remains mostly unaltered during fixation with formaldehyde (Mason and O’Leary, 1991). Even when the quaternary structure is changed by formaldehyde fixation, the secondary structure can remain intact. Formaldehyde does not react with all functional groups in proteins with equal rapidity; it reacts first with lysine and cysteine, and subsequently these amino acids are

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crosslinked to glutamine, asparagine, and arginine. This does not mean that the fixative alters the amino acid sequence of the antigen molecule; the structure of the epitope is maintained even though it may be masked. However, various proteins respond differently to fixation with formaldehyde. It is relevant to point out that aldehyde fixation does not completely abolish the selective permeability of cell membranes (Hayat, 1986). Therefore, it can be inferred that membrane proteins and other proteins are not destroyed by fixation with formaldehyde. The strongest indirect evidence in support of the role of protein crosslinking in creating a barrier to the penetration of antibodies and their reaching to the epitopes emerges from the crosslinking ability of glutaraldehyde. It is known that this dialdehyde introduces extensive protein crosslinks that are mostly irreversible, which significantly inhibits antigenicity (Hayat, 1986, 2000a). This inhibition is due to the crosslinking of protein antigens as well as crosslinking and compacting of proteins surrounding the antigen molecule. Most epitopes in most protein antigens studied so far are located at or near the exposed surface of the antigen molecule. Therefore, compacted proteins surrounding the antigen molecule will prevent the recognition of epitopes by the antibody. The small size of the epitopes makes them susceptible to masking by the surrounding crosslinked proteins.

Comparison of Formaldehyde with Glutaraldehyde
The effect of aldehydes on tissue proteins is exceedingly complex. Numerous factors influence the immunostaining of epitopes, and the fixation of the tissue with aldehyde is the most important parameter. Fixation with an aldehyde results in the crosslinking of proteins, including antigens, resulting in their stabilization, protection, and anchoring in situ. Crosslinking protects the modified antigens from denaturation at elevated temperatures. It is known that the use of high temperatures, for example from a microwave oven, may permanently denature unfixed antigens (epitopes), whereas similar temperatures have a minimal adverse effect on aldehyde-fixed antigens. Studies of unfixed, purified proteins indicate that they show denaturation transitions in the 70–90°C temperature range, whereas such proteins do not exhibit these changes at the same temperature when they have been placed in formaldehyde solutions (Mason and O’Leary, 1991). The absence of phase transition is thought to be due to stabilization of the protein structure by formaldehyde treatment. Glutaraldehyde or formaldehyde alone or together are most commonly used to stabilize proteins. Both have advantages and limitations, as explained below (Hayat, 1986, 1999). Glutaraldehyde molecule is a dialdehyde with two aldehyde moieties, one on each end of a straight hydrocarbon chain.

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The presence of the two aldehyde groups is the reason for its being the most effective protein crosslinking molecule. Glutaraldehyde introduces both intramolecular and intermolecular protein crosslinks that are predominantly irreversible during subsequent processing, including incubations of the tissue. This versatile property results in excellent preservation of the ultrastructure; however, it also has limitations. The dialdehyde produces strong, irreversible protein crosslinks when used in standard concentrations (2–3%). Therefore, it masks most epitopes. In addition and more important, steric hindrance resulting from extensive crosslinking of cellular proteins in general inhibits the antibodies from reaching the intracellular epitopes. Thus, glutaraldehyde is less suitable for light and electron microscopic immunocytochemistry. Some hardy antigens that are less susceptible to the effect of aldehydes, however, can be studied following fixation with glutaraldehyde alone or in combination with formaldehyde for electron microscopic immunocytochemistry. For light microscopic immunohistochemistry and immunocytochemistry, formaldehyde is preferred over glutaraldehyde because it is a monoaldehyde and thus penetrates the tissue more quickly but crosslinks proteins more slowly than glutaraldehyde, a dialdehyde. Formaldehyde does not react with all the functional groups in proteins with equal rapidity. In its reactions with proteins, the first step involves the free amino groups with the formation of amino methylol groups, which then condense with other functional groups such as phenol, imidazole, and indole to form methylene bridges The occurrence of these bridges is considered responsible for the fixation of proteins by formaldehyde under conditions of fixation. The process of masking is progressive, i.e., the longer the fixation, the stronger the crosslinking as well as epitope masking. It is well established that with increasing durations of fixation, antibodies show a nearly linear decrease in immunostaining, indicating a successive masking of epitopes (Werner et al., 1996). Therefore, the duration of fixation should be taken into account while determining the duration of treatment for epitope retrieval. Generally, mild fixation with formaldehyde is preferred. Some epitope types are not affected by standard or prolonged fixation with formaldehyde or a mixture of formaldehyde and glutaraldehyde or, as stated earlier, glutaraldehyde alone. The advantage of using a mixture of formaldehyde (4%) and glutaraldehyde (~0.1 % or a lower concentration) is improved preservation of cell structure. Although formaldehyde is better than many other fixatives for preserving morphological details, it is far inferior to glutaraldehyde. The quality of morphological preservation with glutaraldehyde has been compared with that obtained with formaldehyde (Hayat, 2000a). These studies clearly indicate superior structural preservation with glutaraldehyde. In fact, fixation with formaldehyde is unacceptable for routine electron microscopy, which demands superior structural preservation because of its high resolving power. However, for most electron microscopic immunocytochemical studies, a mixture of formaldehyde and glutaraldehyde is recommended because many types of epitopes are irreversibly masked when the latter alone is used. Although at present formalin is routinely used for the fixation of surgical tissue specimens, the advantage of a mixture of formaldehyde and glutaraldehyde cannot be overemphasized.

Fixation with Formaldehyde
There is no optimal universal fixative for all types of antigens, so the choice of a fixative depends on the type of epitope and the tissue under study. Furthermore, the selection

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of a fixative is always a compromise between the preservation of antigenicity and cell morphology. Although the best fixative for a specific epitope is determined by trial and error or by recommendations from a published study, 4% formaldehyde in 0.1 M buffer (pH 7.2) is recommended for adequately preserving both the antigenicity and cell morphology for immunostaining. Alternatively, 10% neutral-buffered formalin can be used. Standard fixation for 4–6 hr at room temperature is considered a mild and effective fixation. Most of the reactions of this aldehyde with proteins during mild fixation are reversible, and the resultant protein crosslinks for the most part can be weakened and/or broken with treatments such as heating. Unbound formaldehyde is also removed during washing. The reactions of formaldehyde with proteins are influenced by several factors, including its concentration, pH, temperature, and duration of fixation. In general, higher values of these parameters result in increased binding of formaldehyde. The maximum binding occurs at pH 7.5 to 8.0. However, it should be noted that the fixation throughout the tissue block is completed in ~24 hr, but pathological specimens are usually fixed for less than this duration before their further processing. As a result, the tissue is only partially fixed with the aldehyde, and its fixation is completed with the dehydrating ethanol. It means that the tissue is fixed partly by protein crosslinking with the former and partly by coagulation with the latter (Battifora, 1991). Consequently, the specimen may show heterogenicity of immunoreactivity; different areas of the section may exhibit different intensity of staining. Variable staining density in different areas of the tissue block may also be caused by the presence of air bubbles in the vial containing the specimens during fixation. Tissues fixed with coagulating fixatives such as ethanol generally do not benefit by antigen retrieval treatments. Although 10% neutral buffered formalin is the most commonly used fixative and yields satisfactory results, the optimal preservation of certain antigens requires a different fixative. Three examples are given. The immunostaining of transforming growth and thrombomodulin in the human skin has been reported to be superior in the specimens fixed with methanol-Carnoy’s solution (methanol: chloroform: acetic acid, 6:3:1) compared with that in the formalin-fixed specimens (James and Hauer-Jensen, 1999). Optimal duration of fixation is 24 hr. Similarly, the antigenicity of the proteinase-K–resistant form of the prion protein in brain tissue is better preserved in the Carnoy-fixed specimens (Giaccone et al., 2000). Another example is leukocyte antigenicity in various rat tissues, which is better preserved in the Carnoy-fixed specimens (Shetye et al., 1996).

Effect of Prolonged Fixation with Formaldehyde
Why does prolonged fixation with formaldehyde cause progressive loss of immunoreactivity? The reasons are fairly clear. Formaldehyde, being a small molecule penetrates readily and adds onto protein molecules, forming mostly reversible crosslinks. However, the crosslinking occurs slowly because formaldehyde contains only one aldehyde group, and it takes time to align a molecule(s) to accomplish crosslinking. Thus, reversible crosslinks and loosely bound fixative molecules are mostly removed during antigen retrieval processing, resulting in satisfactory immunoreactivity. As the time of fixation increases, additional stronger crosslinkages are formed, which in turn cause progressive loss of immunoreactivity. Such strong crosslinkages may not only alter the

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conformation of the antigen molecule but also create a barrier to antibody access to the epitope. The formation of extensive crosslinkages during prolonged fixation for weeks or months is supported by the observation that such tissue blocks become hard and difficult to section. It is well known that archival tissues are difficult to section. The effect of prolonged fixation with formaldehyde on the antigenicity of the nucleus may differ from that of the cytoplasm. This phenomenon is exemplified by Bcl-2 and Bax, members of the same family of proteins involved in apoptosis regulation; these proteins reside in the cytoplasm as well as in the nucleus. It was recently demonstrated that prolonged fixation with formaldehyde alone irreversibly reduced nuclear or mitotic Bcl-2 immunoreactivity even after heat-mediated antigen retrieval in monolayers of MCF-7 human breast cancer cells (Hoetelmans et al., 2001). Heat treatment, on the other hand, elevated cytoplasmic immunoreactivity of Bcl-2. However, nuclear and mitotic Bcl-2 immunoreactivity was clearly present when these cells were fixed with formaldehyde (3.6%), followed by postfixation with methanol for 10 min at –20°C. Treatment with ice-cold methanol makes the cell membrane permeable, allowing antibody access to intranuclear antigens without protein relocalization. Extensive protein crosslinking with formaldehyde is required for maintenance of intranuclear Bcl-2 immunoreactivity. In contrast to Bcl-2, Bax immunoreactivity was detected in nuclear and cytoplasmic compartments regardless of the duration of formaldehyde fixation used. In light of the aforementioned information, when tissue specimens are exposed to formaldehyde for longer durations due to unavoidable circumstances, immunohistochemical findings should be interpreted with caution, as many tissue antigens could be lost or irreversibly masked.

Formalin Substitute Fixatives
Because formalin poses occupational hazards, a number of ethanol-based fixatives have been introduced, some of which are commercially available. These substitutes for formalin include Notox, Omnifix, Stat Fix, Histochoice, Tissue-Tek, F13, Carnoy, Bouin fluid, and methacarn. Although these fixatives have been recommended for histology laboratories, in comparison with formalin they have limitations. Many of these formalin substitutes are coagulating fixatives which precipitate proteins by breaking hydrogen bonds in the absence of protein crosslinking. This limitation results in inadequate cellular preservation. These substitutes also cause cellular shrinkage and brittleness. The inclusion of polyethylene glycol or acetic acid is thought to minimize these harsh effects (Warmington et al., 2000). Another drawback of some of these formalin substitutes is that they tend to cause an artifactual shift in the intracellular immunoreactivity. Such a shift from the cytoplasm to the nucleus has been demonstrated for growth factor peptides (Bos et al., 2000). The result of such a shift is false nuclear signal dominance or exclusive nuclear staining. This shift is thought to be owing to electrostatic interaction between ligands and DNA or other nuclear components, as fixation (anchoring of cell components) remains incomplete with alcohol-based fixatives. Furthermore, these fixatives make the nuclear membrane permeable, allowing bidirectional movement of ligands between the cytoplasm and the nucleus. In conclusion, the chemistry of formalin fixation is well known, whereas this is not true for the formalin substitute fixatives. Therefore, these substitutes cannot be recommended for immunohistochemistry.

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Another fixative, Kryofix (E. Merck, Darmstadt, Germany) has been recommended as a replacement for formaldehyde in immunohistochemistry by Boon and Kok (1994). Kryofix is a coagulant fixative containing 50% ethyl alcohol and polyethylene glycol (mol. wt. 300). Both ethyl alcohol and polyethylene glycol diffuse rapidly into the tissue, and the tissue fixation is completed in Kryofix in 90 sec with microwave heating. I do not have personal experience with Kryofix.

Fixation Conditions
Some major factors that adversely affect immunostaining are delays in transferring the tissue blocks into the fixative after their surgical removal as well as shorter or longer than optimal duration of fixation. Any delay in the exposure of the tissue to the fixative invites increasing proteolytic degradation of antigenicity. Therefore, if possible, surgical tissues must be placed directly into the fixative immediately after their removal. If delay is unavoidable, the tissue can be refrigerated prior to fixation. It is not uncommon for thick (~5 mm) surgical tissues excised for diagnostic pathology to be underfixed with formalin, especially the core of the tissue block. As an average, fixation in formalin solution for less than 24–48 hr, depending on the size of the tissue block, tends to crosslink only the periphery of the specimen. Under this condition, the core of the tissue block either remains unfixed or fixed by coagulation with the alcohol used subsequently for dehydration. Sections cut from the core tend to show autolysis and inadequate immunostaining, resulting in false-negative staining. Such inadequate staining can be improved by attaching paraffin sections to glass slides and then removing the paraffin with an organic solvent (Eltoum et al., 2001). This treatment is followed by rehydration, buffer rinse, and refixation with formalin. The fixative is removed by rinsing with buffer before staining. If necessary, incompletely fixed, paraffin-embedded tissues can be deparaffinized and refixed and reembedded, risking some damage to cell morphology and immunogenicity. These corrective methods become useful only when additional tissue specimens are not available. Overfixation of specimens may also be encountered, resulting in weak or absent immunostaining, depending on the susceptibility of a specific antigen to the fixative. As already emphasized, prolonged fixation introduces excessive protein crosslinking, which hampers antigen accessibility to the antibodies. In addition, 10% formalin solution contains only 4% formaldehyde, and the remaining components may damage antigens during prolonged fixation. If prolonged fixation has been carried out, immunostaining can be increased by the following steps: robust antigen retrieval by heating, higher antibody concentrations, longer durations of incubations in the reagents, and signal amplification. To determine the optimal increase in heat or protease treatment to counteract the effects of prolonged fixation, three slides can be processed with progressive doubling of the duration of treatment (Werner et al., 2000). The best stained slide of the three is used for interpreting the results of the study. However, some of these approaches may increase the background noise. Overfixation of specimens is also recognized by difficulty in sectioning because of excessive hardness of the tissue. This problem arises when tissues are fixed with formulations containing ethanol, methanol, or acetone. Excessive dehydration with an organic solvent may also cause tissue hardness, especially of small specimens (1–2 mm). Such

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specimens may shatter instead of being sliced during cutting. The increased hardness can be prevented by shortening the duration of dehydration. If the tissue has been excessively hardened, it can be partially corrected by briefly soaking in water (Eltoum et al., 2001). Water tends to penetrate up to 0.5 mm into the tissue block. A large tissue block can be cut into small pieces before soaking in water. Overfixation with protein crosslinking aldehydes can also be partially reversed with washing in water or aqueous buffers.

EFFECT OF HEATING ON FIXATION WITH GLUTARALDEHYDE
No other fixative surpasses glutaraldehyde in preserving cellular details, provided the specimen size is very small. Homogenous, irreversible protein crosslinking is the reason for the superior fixation with this dialdehyde. However, glutaraldehyde penetrates the tissue block rather slowly for three reasons: (1) it is a relatively large molecule, (2) rapid protein crosslinking in the outer layers of the tissue block hinders its deeper penetration, and (3) because fixation is carried out at a slightly alkaline pH, the presence of glutaraldehyde oligomers is promoted, and they do not penetrate the tissue as rapidly as the free monomer dialdehyde. Rapid penetration of the fixative avoids postmortem alteration in the tissue specimen. The speed of glutaraldehyde penetration (diffusion) can be increased at higher temperatures, provided the duration of fixation is short. Diffusion increases exponentially as temperature rises. Diffusion of glutaraldehyde can be increased in the presence of microwave heating. Compared with other heating systems, microwave heating increases the temperature rapidly and uniformly throughout the tissue block. There are several reasons for such acceleration of glutaraldehyde diffusion, which are explained below. Microwave heating causes depolymerization of glutaraldehyde into small monomers, enhancing the fixative penetration as well as acceleration of its reactions with amine groups. Microwave heating also induces changes in the tertiary structure of proteins, exposing reaction sites that remain unexposed at room temperature for reaction with glutaraldehyde (Kok and Boon, 1990). It is also known that microwaves accelerate the diffusion of polar compounds in the presence of concentration gradients. Because both water and glutaraldehyde solutions are polar, they are capable of absorbing energy while being heated with microwaves. Consequently, fixation with dialdehyde in a microwave oven can be accomplished in seconds or minutes for light and electron microscopy. As stated above, monomeric glutaraldehyde diffuses rapidly compared with its polymers. Heating increases absorption at 280 nm, which is the purification index for monomeic glutaraldehyde (Ruijgrok et al., 1990). Monomeric glutaraldehyde diffuses faster than do its oligomers. However, longer durations of heating tend to produce significant amounts of alpha and beta unsaturated polymers, which exhibit absorption at 235 nm. Microwave heating permits the use of glutaraldehyde at very low concentrations, which still yield good ultrastructural preservation and superior antigen preservation. Fixation with glutaraldehyde in a microwave oven also minimizes tissue shrinkage. Moreover, microwave heating prevents some of the artifacts formed during glutaraldehyde fixation. A well-known example is the presence of parathyroid cell variants that are regularly seen in immersion-fixed specimens. These variants can be avoided with fixation by vascular perfusion, however, in certain cases vascular perfusion is not possible.

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A better alternative involves glutaraldehyde fixation by heating in a microwave oven. This approach completely prevents the artifactual parathyroid cell variants (Marti et al., 1987). The fixation in this case is uniformly accelerated throughout the specimen block. Some evidence indicates that microwave heating followed by osmication enhances postfixation with this metal. However, fixation with glutaraldehyde at standard concentrations (2–3%) with or without microwave heating is not recommended for immunohistochemical and immunocytochemical studies, except for localizing epitopes that are resistant to this dialdehyde. The reason for this nonrecommendation is that most epitope types become irretrievable after fixation with glutaraldehyde. Nevertheless, when the quality of ultrastructural preservation has an equal priority with that of immunolabeling, glutaraldehyde can be used at very low concentrations in a mixture containing 2% formaldehyde. For example, cell surface epitopes of rat mast cells were preserved by using this procedure (Jamur et al., 1995). The cells were fixed with a mixture of 2% formaldehyde and 0.05% glutaraldehyde containing 0.025% in 0.1 M cacodylate buffer (pH 7.4) for 4 sec at 100% power in a 550-W microwave oven. As explained in this chapter, glutaraldehyde introduces mostly irreversible protein crosslinks that may alter the conformation of the antigen (epitope) molecule. Such extensive crosslinkages become a barrier to the antibody penetration, and thus its accessibility to the epitope is hindered. This impediment becomes a serious problem when monoclonal antibodies specific for only one epitope type are used and/or when epitopes are not located at the surface of the antigen molecule. As explained earlier, large polymers of glutaraldehyde are formed in the outer layers of the tissue block, which impede further penetration of the fixative into the core of the tissue. Such an impediment is due to steric hindrance and/or formation of nucleation sites to which fixative molecules may attach. The latter will consume large quantities of glutaraldehyde, depleting its concentration in the solution. The net result will be uneven fixation of the tissue block. The uneven fixation, however, can be avoided by providing conditions that initially allow an adequate penetration of glutaraldehyde throughout the tissue block, followed by the formation of crosslinks and polymers. This can be accomplished by presoaking the specimen in a low concentration (0.5%) of glutaraldehyde at 0–4°C, followed by microwave heating of the fixative at 45°C (Ruijgrok et al., 1993). The durations of these two steps are determined by trial and error. The initial cold temperature slows the formation of polymers in the outer cell layers of the tissue, allowing glutaraldehyde penetration. An instant homogenous heating in a microwave oven facilitates uniform protein crosslinking throughout the tissue block after the fixative has fully penetrated. Other types of heating do not impart an even rise in temperature throughout the tissue block.

MICROWAVE HEAT–ASSISTED FIXATION WITH OSMIUM TETROXIDE
Standard chemical fixation fails to preserve extracellular materials. In contrast, heating or the high-pressure freezing-freeze-substitution technique is able to preserve such materials (Eggli and Graber, 1994). The former technique is simpler

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and cheaper and can be completed in a conventional microwave oven with a maximal power output of 650 W or higher. A water load (200 ml in a beaker), placed in a rear corner of the oven, serves as a damper to increase heating times to 10 sec or longer. Tissue specimens (e.g., nervous, ocular, and skin tissues) are fixed for 2 min at room temperature with 2% in 0.2 M sodium cacodylate buffer (pH 7.2, 450 mOsm). They are transferred into small glass vials containing 10 ml of the fixative. The vials are placed in the center of the microwave oven and heated at maximal power output and at a frequency of 2,450 MHz until the temperature between 43°C and 40°C is attained (~12 sec). The specimens are removed from the oven and kept at room temperature for 10 min prior to washing in 0.1 M buffer and embedding in an epoxy resin. The results of this procedure are shown in Fig. 3.1.

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ROLE OF MICROWAVE HEATING IN ENZYME CYTOCHEMISTRY
Typically, residual enzyme activity is localized at the subcellular level after fixation with an aldehyde followed by incubations. Aldehydes, especially glutaraldehyde, denature enzyme molecules to various degrees. Lengthy incubations under nonphysiological conditions may cause the loss of structural details. To improve preservation of both the enzymatic activity and the ultrastructure, incubation durations can be shortened under microwave heating (Rassner et al., 1997). The advantage of incubation under microwave heating becomes apparent when one considers that it allows incubation of tissue specimens, whereas conventional incubation requires tissue slices of 0.1 mm. Tissue specimens are fixed for 16 hr with a mixture of 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) containing 0.06% calcium chloride. After rinsing in buffer, the specimens are placed in 10-ml snap-cap glass vials containing 2 ml of incubation medium. The vials are capped to avoid contamination of the incubation medium from the water bath. They are placed into a staining jar (3 × 3.5 × 2.5 inches) which has been filled with 150 ml of tap water at 22°C. The microwave incubations are carried out twice for 30 sec each in a microwave oven at the highest power setting (900 W at 2,450 MHz, with two water loads 250 ml each) placed in the rear of the oven. The temperature in the oven is not allowed to exceed 40°C. The water of the water bath is changed between the two 30-sec pulses. The temperature of the water bath rises during microwave heating from 22°C to 40°C. The staining jar is removed from the oven, and the incubation is continued for an additional 30 min at 37°C. After washing with distilled water, the specimens are postfixed with a mixture of 1% and 1.5% potassium ferrocyanide in 0.1 M cacodylate buffer (pH 7.2) for 90 min in the dark at room temperature. The specimens are rinsed in buffer and then immersed in 2% aqueous uranyl acetate. Detailed methods for achieving accelerated visualization of acetylcholinesterase activity at motor endplates using microwave heating have been presented by Petrali and Mills (2001).

Fixation for Enzyme Cytochemistry Using Microwave at Relatively Low Temperature
The activity of some enzymes can be preserved by tissue fixation with aldehydes in a microwave oven at a low temperature for electron microscopy. This procedure has been used for observing cytochrome oxidase in mitochondria in hamster submandibular gland tissue (Moriguchi et al., 1999). The activity of this enzyme has also been studied in the isolated mitochondria of this tissue, using the same method (Moriguchi et al., 1998). This procedure also has been employed for confocal laser scanning microscopy of enzymatic activity (Moriguchi et al., 1999). The following protocol was used for processing the tissue for electron microscopy. The microwave processor is fitted with a thermometer, timer, stirrer, and power level controller (150 W to 400 W) (MI-77, Azumaya Company, Tokyo, Japan). One glass Petri dish with 70-ml capacity containing 50 ml of a mixture of 2% paraformaldehyde and 0.5% glutaraldehyde containing 45 mg/ml sucrose in 0.1 M PBS (pH 7.4) is placed in a large Petri dish containing 250 ml of chilled water in the processor. The specimens are heated in a microwave oven for 10 min at ~4°C at a low energy level of 150 W.

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CRYOPRESERVATION IN THE PRESENCE OF MICROWAVE HEATING
Cryoinjury to the specimen is caused directly by extra- or intracellular ice crystal formation as well as by ice-induced solution effects during cryopreservation. Ice crystals seriously deform cell components. Another disadvantage of the formation of ice crystals near the specimen surface is slowing the cooling rate in areas below the surface because their thermal conductivity is about half that of solid water in a noncrystalline state. Furthermore, ice crystal formation is accompanied by the generation of latent heat, which also slows down the freezing rate. Apparently, cryoinjury can be avoided by eliminating ice crystal formation and vitrifying the specimen. Vitreous state can be achieved by ultrarapid cooling (> ) or using high concentrations of a cryoprotectant. However, the former is difficult to attain, and the latter tends to cause chemical toxicity and high osmotic stress. Because biological specimens possess low thermal conductivity and high thermal capacity, ultrarapid cooling can be obtained only for very small specimens. The above-mentioned difficulties in obtaining ultrarapid freezing can be minimized in the presence of microwave heating. This treatment suppresses ice crystal formation near the specimen surface, thereby extending the depth of good freezing from the specimen surface. Another advantage is better reproducibility of results because the state of water near the specimen surface is under control with microwave heating. Two mechanisms responsible for decreased rate of ice crystal growth are suggested. It is possible that the electric field component of electromagnetic radiation interacts with dipolar water molecules, disrupting the ice nucleation phenomenon (Hanyu et al., 1992). In other words, microwave heating reduces the size and number of ice crystal nucleation centers near the specimen surface. An alternative explanation is based on microwave radiation interfering with the kinetic processes of ice crystal growth (Jackson et al., 1997). For an ice crystal to form and grow, each water molecule must have an appropriate spatial orientation, position, and energy. Rapid ice crystal growth requires the molecular clusters to share edges and faces with the ice lattice without the induction of mutual strains. The torques produced by a microwave field can increase the number of available isomeric configurations, reducing the likelihood of a cluster of molecules having a configuration suited to integrating into a crystal lattice. Further development in the application of microwave heating to vitrification of biological specimens is awaited. Two apparatuses have been constructed for achieving ultrarapid freezing in the presence of microwave heating (Hanyu et al., 1992; Jackson et al., 1997). Microwave treatment can be employed with or without a cryoprotectant. According to one method, microwave heating is used at 2.45 GHz for a short duration (50 msec) immediately before and during tissue contact with the surface of a copper block cooled with liquid nitrogen (Hanyu et al., 1992). The ultrastructure is well preserved to a depth of from the contact surface, which is comparable to the depth obtained by the metal contact method using liquid helium in the absence of microwave heating.

PARAFFIN EMBEDDING
For routine immunohistochemestry, surgical and other tissues are embedded in paraffin which is a mixture of hydrocarbons. Automated paraffin tissue processors are commercially available that customize the schedule to meet specific needs. However, tissues of

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various types and sizes as well as the particular study objective require optimal conditions of dehydration, infiltration, and embedding in paraffin. Chemical and physical changes occur in specimens during these treatments, affecting the sectioning and immunostaining qualities. Longer than optimal durations of these steps is a common habit, resulting in hard and brittle tissues that are difficult to section. It should be noted that additional fixation occurs during dehydration, accompanied by antigen masking and lipid dissolution. After fixation, a series of ethyl alcohol (a water-miscible solvent) of ascending concentrations is used to remove water from the tissue. Free water from the tissue is easily removed by diffusion. Water attached to the tissue by hydrogen bonds is also replaced by ethyl alcohol of higher concentrations. The efficacy of a solvent depends on its hydrogen bonding strength and molecular weight (Wynnchuk, 1993). Higher temperatures, vacuum, and microwave heating expedite the speed of dehydration, allowing shorter durations of dehydration. Xylene (an aromatic hydrocarbon) is used to replace ethyl alcohol from the tissue before infiltration with paraffin. Xylene is miscible with ethyl alcohol and paraffin. Xylene is called a clearing agent because it has a refractive index similar to that of proteins and thus renders tissue more or less transparent. It is generally satisfactory when the tissue blocks are not thicker than 3–4 mm. Xylene must be completely removed with paraffin, otherwise tissue will not section. Excessive exposure to xylene causes further denaturation of tissue proteins, causing difficulties in sectioning. Some lipid extraction also occurs in the presence of xylene. The treatments mentioned above to expedite the diffusion of ethyl alcohol also speed up the penetration of xylene. Xylene is also used between ethyl alcohol and mounting sections with resinous mounting medium after staining. The volatility and inflammability of xylene render it potentially dangerous. It must be used in a fumehood. While tissue is in xylene, gradual infiltration with paraffin is carried out. For tissues of a small size, 2 to 3 hr of paraffin infiltration is adequate. For large tissues (5–10 mm), overnight infiltration is required. The temperature during infiltration must not be higher than 4° above the melting point of paraffin (54–58°C). Vacuum embedding can be carried out to remove air bubbles from the tissue and rapidly replace the clearing agent with paraffin. This approach is especially desirable for aircontaining tissues such as lung or hard tissues such as fibrous or scar tissues. The vacuum should not exceed 400–500 mm of mercury to avoid damage to the tissue. Paraffin blocks are trimmed with a scalpel, a razor blade, or a hot spatula and mounted on wooden or fiber blocks. While being sectioned, longitudinal block edges must be parallel to the knife edge to obtain a ribbon. Paraffin sections of any thickness show compression, which is usually relieved when they are floated on a glass slide and dried. It should be noted that the melting point and crystalline structure of paraffin influence the section quality. Paraffin of a lower melting point is less brittle when solidified; however, it tends to show more compression during sectioning. On the other hand, paraffin of a higher melting point provides a better support for hard specimens. Paraffin of a smaller crystalline structure adheres closely to the cell components in the embedded tissue, providing good support for sectioning. Paraffin sections of a larger crystalline structure show more pronounced curvature, which is difficult to flatten after sectioning. To obtain crystals of a small size, paraffin should be cooled rapidly. Deeper layers of the tissue block contain larger and looser crystals, resulting in poor quality of sections in these layers.

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Paraffin Embedding in Microwave Oven
Recently, a new type of microwave oven (HFX-800, Meditest, Illatos ut 9, 1097 Budapest, Hungary), combining microwave heating and vacuum, was introduced (Kovacs et al., 1996). The oven is reported to allow fixation, dehydration, paraffin embedding, and section staining. The histoprocessing is completed in depending on the thickness of the tissue block. Decalcification of the bone specimen can also be carried out in this oven. The power level can be switched from 10 to 650 W by the chosen cycle time. The temperature can be preset and subsequently controlled automatically between 0 and 120°C. Vacuum is generated by a built-in vacuum pump (operating at 12 V), producing 0.0–0.3 bar vacuum. The vacuum level is indicated by an automatic meter. Since the oven provides a sealed and ventilated system, the evaporation of formalin, ethanol, isopropanol, and other reagents does not affect the operator. Vapors and fumes are extracted by continuous ventilation. The oven weighs about 21 kg. Its use is awaited.

Paraffin Embedding in Vacuum-Microwave Oven
Vacuum combined with microwaving has been tried for embedding the tissue in paraffin, using Milestone’s MicroMED LAVIS-1000 machine (Marani et al., 1996; Bosch et al., 1996). The advantage of this system is that microwaves travel with ease through a vacuum, whereas conventional heating under vacuum is difficult. This machine provides pressure reaching 100 hPa, and the microwave oven attains a maximum power of 1,000 W; its cycle time can be adjusted between 0.1 and 0.5 sec. The machine is equipped with an infrared temperature probe which allows temperature control from outside the unit. To obtain satisfactory results, coordination of temperature with vacuum is necessary. Paraffin embedding is carried out in a stepwise descending series: 700 hPa, 500 hPa, 300 hPa, and 100 hPa. A too-rapid lowering of the pressure is damaging to tissue morphology. The temperature during dehydration with isopropanol should not exceed 60°C. Further improvements of this system are awaited.

Microtomy of Paraffin-Embedded Tissues
Commercial paraffin is a mixture of a straight chain of hydrocarbons that contain additives. Both the melting and the plastic points of paraffin are related to the sectioning properties. The plastic point occurs ~10°C below melting point. The role of the melting point becomes apparent when one considers that the higher-melting-point hydrocarbons crystallize first as flat plates that accumulate on one another as successively lower-meltingpoint hydrocarbons crystallize (Allison, 1998). These dynamic processes force the plates to curl and roll, giving rise to needle-shaped crystals. Needle-shaped crystals are considered ideal for microtomy. Thus the proportion of plates and microcrystals depends on the proportion of high-melting-point and low-melting-point hydrocarbons in the paraffin. The shape and the size of crystals are influenced by the nature of cell and tissue structures as the molten paraffin infiltrates and solidifies in the tissue spaces.

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Two types of forces are exerted during paraffin sectioning: flow shearing and pointto-point shearing (Allison, 1998). Flow shearing proceeds ahead of the cutting edge, resulting in smooth sections. In contrast, point-to-point shearing travels through the path of least resistance ahead of the cutting edge, producing a section of uneven thickness. Paraffin contains additives that minimize the point-to-point shearing and reduce the plastic flow. Additives are synthetic polymers that improve the consistency of paraffin by filling the spaces among paraffin crystals in the tissue. Cut a paraffin block containing one tissue specimen with a razor blade, and mount it to a support stub. Trim the block manually with a razor blade or on an automatic trimming microtome to a rectangular or trapezoidal cutting face. The size of the block face is determined by the objective of the study and the size of the tissue specimen. The upper and lower edges of the block facing the knife cutting edge should be parallel to each other to obtain a ribbon, if required. Mount and orient the block on the microtome, so that its longer edge is parallel to the cutting edge. A steel or glass knife can be used. Cut sections ( thick) on a rotary microtome, which usually has an automatic advance mechanism that can be set to advance the specimen block the desired distance toward the knife with each stroke. Manual or motorized rotary microtomes are commercially available (Triangle Biomedical Sciences, Durham, NC; Sakura Finetek, Torrance, CA). A microtome with automated specimen approach, trimming, and sectioning is also available (Leica Microsystems, Deerfield, IL). Float the sections on water or 4% formalin on a glass slide and heat for 10–15 min at ~40°C on a warming plate to remove the compression. Remove the liquid with a fine pipette and dry overnight in an oven at ~40°C to ensure section adherence to the slide. Drying can also be accomplished in 15 min at ~40°C in a microwave oven. Note: Sectioning will be adversely affected if tissue infiltration with paraffin is incomplete. A too-shallow or too-steep bevel angle of the steel knife relative to the tissue block face will result in section compression and chatter, respectively. The optimal cutting angle is 4°. Section adhesion to the glass slide can be ensured by coating the slide with polylysine ( to 1 mg/ml) in 10 mM Tris (pH 8.0). Alternatively, coated slides are commercially available (Probe-On-Plus slides from Fisher Scientific). Make sure that the tissue specimen is firmly mounted to the stub and the latter to the microtome. Also, the paraffin block should be fairly cold at the time of sectioning. Low humidity in the vicinity of the microtome tends to result in static electricity, which makes it difficult to separate the section from the block face after cutting. For additional details, see Ruzin (1999).

Silanting of Glass Slides

Detachment of tissue sections from glass slides during processing for immunohistology is not uncommon. Such detachment can be avoided by preparing silanted slides as follows (Miksys, 1999).
1. 2. 3. 4.

Wash slides in common household detergent. Rinse in running tap water for 10–15 min. Rinse in distilled water. Rinse in 100% acetone for 5 min.

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5. Coat with 2% Aplex (3-aminopropyltriethoxysilane) (Sigma) in 100% acetone for 5 min. 6. Rinse in lightly warm running tap water for 2 min. 7. Rinse in distilled water. 8. Dry at ~40°C in a dust-free area. 9. Store at room temperature up to 1 month or at –20°C for several months. Freshly coated slides are preferred.

Vacuum-Assisted Microwave Heating
A vacuum–microwave combination has been used for processing tissues for light microscopy (Kok and Boon, 1996), transmission electron microscopy of animal tissues (Giberson, 2001) and botanical specimens (Russin and Trivett, 2001), and scanning electron microscopy of human lymphocytes (Demaree, 2001). The vacuum-microwave heating method is especially useful for processing botanical tissues because these specimens possess physical characteristics that hamper easy penetration of reagents; these characteristics include cell wall, vacuoles, plastids, and intercellular spaces. Secondary cell walls may contain cutin, suberin, and lignin, which are hydrophobic. These waxy substances limit the evaporation of water from the tissue and resist the penetration of reagents. The presence of air in the intercellular spaces creates a barrier to fixative penetration. These impediments to reagent penetration and action of fixatives can be significantly reduced by using vacuum–microwave heating. For details of this methodology, see Russin and Trivett (2001).

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Chapter 4

Factors Affecting Antigen Retrieval

Many factors influence antigen retrieval, including fixation, heating, retrieval fluid, and antibodies.

FIXATION
Fixation is the most important factor affecting antigen retrieval. Type of fixative and duration and temperature of fixation are all important. Many varieties of epitopes have been retrieved with various degrees of success in tissues fixed with formalin, methanol, methacarn, or Bouin’s fixative; buffered 10% formalin containing 3.7–4.0% formaldehyde is the most commonly used. Although fixation with paraformaldehyde or glutaraldehyde better preserves cell morphology because of stronger, and more rapid and more extensive protein crosslinking, antigen unmasking becomes difficult. The use of formalin has become a matter of habit and convenience, especially in pathology laboratories; it is also inexpensive. To improve the preservation of cell morphology, it is recommended that a mixture of formalin (or paraformaldehyde) and glutaraldehyde (0.05–0.5%) be tried. Such mixtures are routinely employed for immunocytochemical studies with the electron microscope. It is known that some types of antigens are resistant to fixation with low concentrations of glutaraldehyde. Preembedding immunocytochemistry by the avidin-biotin method, which avoids fixative effects, has been successfully applied for identifying peptide or protein antigens in the brain tissue fixed with glutaraldehyde (Mrini et al., 1995). The effect of fixation on antigenicity is complex. With the exception of a minority of antigen types (e.g., PCNA nuclear protein) that are formalin resistant to various degrees, most antigens are sensitive to the concentration of the fixative and the duration of fixation. Although 10% formalin is the usual fixative, it is inadequate for preserving some types of antigens that are fixative resistant. It has been demonstrated histochemically, for example, that PCNA nuclear protein antigenicity is preserved much better with 20% formalin-PBS than with 10% of the same fixative (Muñoz de Toro de Luque and Luque, 1995). The reason is that the protein antigen is partially extracted with the lower fixative
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concentration. In this respect, it should be noted that some types of antigenicity, such as PCNA, require quantitative measurements for assessment of clinical significance, as its low levels may be present in quiescent cells. Is antigenicity affected by factors other than fixation? Yes. Although fixation is the most important factor in tissue processing for immunohistochemistry, other factors tend to affect immunorecognition of antigens. These factors include the interval between removal of the tissue from the human or animal and fixation; the method of excising the tissue from the body (mechanical damage); the technique of cutting sections of the tissue embedded in paraffin or resin; the procedures for removing section compression, folds, or bubbles and attaching it to the glass slide; the interval between cutting sections and immunostaining (storage or without storage of slides prior to staining); and other immunostaining details. Folds and bubbles hinder section adhesion to the slide, and they may also show 3,3-diaminobenzidinetetrachloride (DAB) precipitation. Bubbles under sections may appear as brown spots on immunostained sections (Grizzle et al., 2001). Paraffin sections ( thick) adhere tenaciously to glass slides by heating overnight at 65°C. The use of a PAP pen or other means to demarcate the tissue to aid in staining is also a variable. Awareness of the above-mentioned variables should prevent erroneously attributing them to problems with fixation. Tissue specimens ideally should be placed in the fixative immediately after their removal from the body. This problem arises in studies of human tissues, for their immediate fixation is usually not feasible. If immediate fixation is not possible, the tissue must be kept cool and moist by covering it with a piece of cloth soaked in sterile, cold saline for not more than 20–30 min. During this time the specimen should not contact any dry and absorbent object such as paper, a paper towel, or gauze. To keep the paper trail of the specimen (source, time, place of collection, etc.) is no less important. Note that even human tissues fixed immediately after their removal from the body may undergo cellular changes because usually the vascular supply is terminated before the tissue is surgically removed. During this duration (~1 hr) the tissue remains at body temperature, at which the activity of digestive enzymes continues, damaging the cellular structures (Grizzle et al., 2001). Chemical fixation is not the only factor that causes loss or irreversible masking of antigens. Treatments such as dehydration and embedding following fixation also play a role in the loss of immunorecognition of antigens. Absolute ethanol and xylene must not contain traces of water, and the water bath should be very free from contaminants such as bacteria, fungi, dust, and dirt. Once the sections are contaminated, they cannot be decontaminated. According to Watanabe et al. (1996), the antigen preservation test (Riederer, 1989) showed that immunostaining intensity, for example of decreased during fixation with paraformaldehyde but did not decrease during washing and immunostaining. The proportionate decrease in intensity due to fixation was almost constant even when the amount of the antigen differed in the sections. They concluded that the decrease in immunostaining intensity was related to a proportional decrease in antibody binding due to masking of antigens during fixation.

DENATURATION
On the basis of antigen retrieval obtained with protein denaturing agents, it has been proposed that in certain cases antibodies recognize denatured but not native antigens.

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However, this proposition seems untenable, given the requirement of a specific amino acid sequence for an epitope, as well as a specific conformation of antibody molecule, in order for antigen-antibody binding to occur. How could an antibody react with a completely denatured antigen, when the former is usually generated using the native form of the latter? For example, antibodies generated against selected regions (N-terminal fragment or C-terminal region) of corresponding antigens recognize predominantly similar undenatured regions, unless these regions of the antigen are masked by other regions of the antigen and/or by some surrounding components. The proposition that antibodies recognize certain antigens only after the latter have been denatured is true only when the epitope is unmasked and remains undenatured following antigen denaturation. In other words, a denatured epitope cannot be recognized by the antibody if the amino acid composition of the epitope peptide and/or its linear amino acid sequence is altered or damaged. The reaction between the antigen and the antibody is dependent on the conformation of the former. However, the presence of an intact threedimensional folded antigen structure may not be necessary in certain cases for antibody binding. Denaturation or unfolding of certain antigen molecules may be necessary to unmask the epitope that is buried in the interior of the folded antigen structure (personal communication, Dennis Brown). It is likely that most antigen molecules form multiprotein complexes, resulting in masking of epitopes by surrounding proteins. The epitope masking becomes more serious when these complexes are crosslinked with formaldehyde. Such masked epitopes can be recognized by the antibody only when exposed by breaking crosslinks and denaturing surrounding cell components. If this is so, denaturing treatments cause breakdown of reversible crosslinks introduced by formaldehyde and denaturation of surrounding cell components, enabling the antibodies to recognize the native, uncrosslinked or partially crosslinked undenatured structure of the reactive epitope molecule. It means that denaturing treatments do not denature antigen per se but denature multiprotein complexes of which the antigen is a part. It may be that imprecise nomenclature has given rise to confusion in this field. It should also be noted that denaturing agents make cells permeable, facilitating antibody penetration. Moreover, these agents are used usually in combination with microwave heating. Therefore, the role of these agents in epitope retrieval needs to be explained in the context of their role in cell permeabilization as well as of the influence of elevated temperatures.

HEATING
Different heating methods, including microwaving, autoclaving, pressure cooking, microwaving combined with pressure cooking, steam heating, and water bath heating, have been employed for antigen retrieval with various degrees of success. However, microwave heating, introduced by Shi et al. (1991), is most commonly used for retrieving a wide range of masked antigens in formalin-fixed and paraffin-embedded tissues. Heating at a high temperature (100°C) for a short duration (10 min) gives better results than those achieved with a comparatively low temperature for a longer time. However, there are some exceptions. According to Evers and Uylings (1994a), the immunostaining of SMI32 obtained at 90°C was superior to that achieved at full power heating.

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pH
Another important factor in achieving optimal antigen retrieval is the pH of the retrieval solution. It is thought that the pH is more important than the constituents of the retrieval fluid (Shi et al., 1995a). There is, however, no universally optimal pH for a retrieval fluid. The retrieval of most types of antigens requires a specific pH, although retrieval of a few antigens can be achieved over a wide range of pH levels; for example, AE1 and NSE (cytoplasmic antigens), PCNA (nuclear antigen), and L26 and EMA (cell surface antigens) can be retrieved at pH levels of 1.0–10.0 (Shi et al., 1995a). On the other hand, following retrieval at pH 3–6, some antigens (e.g., estrogen receptor) display a marked decrease in the immunostaining, while still other antigens such as cytoplasmic HMB 45 show weak or negative staining after retrieval at pH 1–2 but excellent results in the high pH range (Shi et al., 1995a). Some antigens can be retrieved only at a low pH. These types are exemplified by thrombospondin and SMI-32 (neurofilament protein), which require pH levels of 1–2 and 2.5, respectively (Grossfeld et al., 1996; Evers and Uylings, 1994a). However, pH levels lower than 3.0 can severely damage tissue morphology. Low pH levels can also alter the localization of some cytoplasmic antigens, resulting in false-positive staining of the nucleus. For example, an antibody (UCHL1) to T cell antigen is effective at pH 6.0 but results in the staining of every nucleus at pH 2.0 (personal communication, H.Y. Lan). In summary, the use of sodium citrate buffer at pH 6.0 increases the intensity and extent of immunostaining of a wide variety of tissue antigens, whereas Tris-HCl buffer may yield better results for some antigens at pH 10.0. On the other hand, low-pH antigen retrieval fluids are necessary for some antigens such as thrombospondin. For previously unexamined antigens, a test battery based on three pH values (low, middle, and high) should be carried out to establish an optimal protocol (Shi et al., 1996a).

MOLARITY
The concentration of antigen retrieval fluid is often less important than temperature, duration of heating, and pH in achieving optimal antigen retrieval. For example, sodium citrate buffer is effective at molarities ranging from 0.01 to 0.5. However, in the case of another antigen retrieval solution, ammonium chloride, 0.5%, 1%, 2%, and 4% solutions were tested, and 4% concentration yielded the best immunostaining of vimentin in archival paraffin sections (Suurmeijer and Boon, 1993a). Ammonium chloride solutions are weakly acidic (pH 3–4). According to Bruno et al. (1992) and Muñoz de Toro de Luque and Luque (1995) minor changes in ionic strength affect the PCNA nuclear protein antigenicity involved in DNA synthesis. If enzyme digestion methods are used, the optimal concentration of the enzyme (e.g., protease) must be applied. Unlike sodium citrate buffer, enzyme solutions cannot be used at a range of concentrations. Note that the concentration of the diluent used for primary monoclonal antibodies does affect the specificity and intensity of immunostaining (see page 82). It should be noted that the concentration of the antibody also affects the specificity and intensity of immunostaining (see page 80).

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ANTIGEN RETRIEVAL FLUIDS
Several antigen retrieval fluids are in use, all having been reported to efficiently mediate antigen retrieval. A fluid applicable to all antigens is not available. The main reason is the enormous variety of chemical structure of not only antigens but also epitopes of any one antigen. In fact, the chemical nature of the epitope plays a key role in the effectiveness of an antigen retrieval fluid. In other words, the tissue, cell, and antigen types determine the retrieval fluid. This is substantiated by the fact that generally each type of epitope determines the fluid type conducive to its maximal retrieval. A few examples follow. Two antigen retrieval fluids, sodium citrate buffer (0.1 M, pH 6.0) and glycine-HCl buffer (0.05 M, pH 3.5) containing 0.01% EDTA, were compared for their effectiveness in unmasking a wide variety of antigens (Imam et al., 1995). Glycine-HCl buffer-EDTA yielded stronger immunostaining of p53, androgen, estrogen, progesterone, and Ki-67, whereas sodium citrate buffer produced superior immunostaining of vimentin and leukocyte antigens. PCNA was unmasked equally well with either of the two antigen retrieval buffers, while the two buffers were ineffective in retrieving antigens such as prostatic acid phosphatase and pan-keratin. According to another study, compared with sodium citrate buffer, Tris-HCl buffer (pH 9.5) containing 5% urea yielded more intense staining of Ki-67 in mouse lung tumors (Ito et al., 1998). However, low background staining is likely when using the latter antigen retrieval buffer. Certain other types of antigens require a combination of antigen retrieval fluids or systems for their optimal retrieval. Methods using such combinations are given in this volume. Comparative effects of antigen retrieval systems on antigens are summarized in Chapter 6. In addition to the chemical structure of antigens, a number of other factors, including pH, heating temperature, molarity, and the chemical composition of the retrieval fluid, are considered for selecting the optimal retrieval fluid. Optimal immunostaining of a given antigen requires an antigen retrieval fluid of a specific pH. Note that optimal antigen retrieval requires an optimal fixation procedure. Although the exact mode of action of antigen retrieval fluids is not known, their salts may modify the hydrophobicity of polypeptide chains, affecting the conformation of protein molecules. The major effect of the salts is to mediate high temperature effects. However, this mechanism does not explain the mode of action of nonbuffer fluids (e.g., water) used for antigen retrieval. Excellent p53 immunostaining in breast tumors has been achieved by heating the sections in water for 15 min at 50°C (Katoh and Breier, 1994). Heating is carried out in a microwave oven or in a water bath. Antigen retrieval can occur under both acidic and alkaline conditions, depending on the type of antigen involved. The mechanisms involved in the antigen retrieval at different pH values are not known. Three commonly used antigen retrieval fluids are 0.01 M sodium citrate buffer (pH 6.0), 0.01 M Tris-HCl buffer (pH 1.0) or 0.1 M Tris-HCl (pH 10.0), and 0.05 M glycine-HCl buffer (pH 3.6). The latter can be used with or without 0.01% EDTA depending upon the antigen type. Taylor et al. (1996b) recommend 0.1 M Tris-HCl buffer (pH 9.5) containing 5% urea. These fluids provide strongly alkaline or acid environments and are effective for antigen retrieval in tissues which have been either mildly fixed or overfixed with formalin. These recommendations are based on the successful immunostaining of a wide variety of antigen-antibody complexes. For most clinical applications, 0.01 M sodium citrate buffer (pH 6.0) is recommended.

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Only if sodium citrate buffer or Tris-HCl buffer fail to yield satisfactory retrieval should fluids containing substances such as EDTA, EGTA, enzymes, metal salts, periodic acid, or urea be tried. Consider the immunostaining of parvalbumin, calbindin, and MAP, which has been found to be best accomplished using 4% aluminum chloride (Evers and Uylings, 1994b). However, in this study the antigen retrieval effect of the metal solution was compared only with that of distilled water and zinc sulfate; sodium citrate buffer was not tested. Since free-floating vibratome sections of the human brain tissue underwent severe wrinkling during microwave heating, thick tissue slices (0.5 cm) were placed in 4% aluminum chloride solution and heated in a microwave oven for 10 min, followed by standard immunocytochemical staining of semithin sections Another advantage of pretreatment is that the brain tissue hardens, facilitating easier sectioning. Fluids other than standard sodium citrate and Tris-HCl buffers are also preferred in some other cases. An example is the retrieval of Bcl-2 antigen (oncoprotein), which is best achieved by hydrated autoclaving of sections placed in deionized water (Umemura et al., 1995). Immunostaining of neurofilament proteins, proliferating cell nucleus antigen (PCNA), retinal S-antigen, and glial fibrillary acidic protein (GFAP) has been obtained by using distilled water as the antigen retrieval fluid in a microwave oven (Yachnis and Trojanowski, 1994). However, heating in water in a microwave oven is not generally recommended. Neurofilament proteins in archival tissues have been immunostained after employing a saturated solution of lead thiocyanate (Yachnis and Trojanowski, 1994). Zinc sulfate has been used for retrieving vimentin and prostate-specific antigen (Wieczorek et al., 1997). Cesium chloride (5.7 M) has also been employed for antigen retrieval. However, such metal salt solutions are not recommended because they are toxic. Target unmasking fluid (TUF) was developed by van den Berg et al. (1993) for routine immunohistochemistry and is commercially available (Signet Lab, Delham, MA, or Kreatech Biotechnology, Amsterdam). Periodic acid (0.5%) has also been used as an antigen retrieval fluid (Xue et al., 1998). Another type of antigen retrieval fluid is EDTA of pH 8.0 (Morgan et al., 1994; Pileri et al., 1997), which has been stated to be effective irrespective of the location of the target molecule (intranuclear, intracytoplasmic, or membrane-bound). Comparative studies by Ehara et al. (1996) also indicate that EDTA (0.15 M, pH 6.0) yields stronger immunostaining of steroid hormone receptors than that obtained with sodium citrate buffer (0.01 M, pH 6.0). However, the preservation of cell morphology is superior when citrate buffer is used. Urea (3 M), formic acid, and guanidine solutions have also been employed for antigen retrieval. When urea is used in an autoclave or a pressure cooker, it has the disadvantage of yielding false-negative results or background staining (Shi et al., 1996b). In another study, a saturated solution of dimedone was applied for antigen retrieval (Shi et al., 1996b). Recently, it was reported that the addition of calcium chloride to the antigen retrieval fluid of a low pH improved the preservation of tissue morphology (Morgan et al., 1997a,b). Boric acid (0.2 M, pH 7.0) in conjunction with low-temperature, heat-mediated antigen retrieval technique has been successfully used as the antigen retrieval fluid for estrogen receptors on freshly cut sections of breast tissue (Peston and Shousha, 1998). Boric acid is also very effective in antigen retrieval on the archival hematoxylin-eosin-stained lymphoid sections on coated or uncoated slides, using conventional heat-mediated antigen retrieval method (Biddolph and Jones, 1999). Lymphoid sections tend to dislodge from the coated or uncoated slides in the presence of sodium citrate buffer during antigen retrieval.

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The above discussion indicates that although 0.01 M sodium citrate buffer (pH 6.0) is commonly used, it is not a universally ideal antigen retrieval fluid for all types of tissues and antigens. If published information is not available with regard to the best antigen retrieval fluid for the antigen under study, the ideal retrieval fluid for each type of epitope must be determined by trial and error. The following four antigen retrieval fluids are commercially available (BioGenex, San Ramon, CA). The approximate pH indicated below is valid at the time of manufacture; the pH of the fluid may change during storage.
1. 2. 3. 4.

Antigen Retrieval Citra Microwave Solution used at pH 6.0. Antigen Retrieval Citra Plus Microwave Solution used at pH 6.1. Antigen Retrieval Glyca Microwave Solution used at pH 3.5. Antigen Retrieval AR-10 Microwave Solution used at pH 10.5.

Other commercial sources for antigen retrieval fluids are:
1. Dako TRS, Dako Corporation, 6392 Via Real, Carpinteria, CA 93013 HIER buffer, Ventana Medical Systems, Tucson, AZ 2. Target Unmasking Fluid (TUF*), Monosan (Sanbio), Fronstraat 2A, Postbus 540, AM Uden, NL-5402, The Netherlands; Serotec Ltd., 22 Bankside, Station Approach, Kindlington, Oxford, Oxon OX5 1JE, U.K.

Glycerin as Antigen Retrieval Fluid
When other antigen retrieval methods fail, antigen retrieval can be accomplished in 90% glycerin solution using a hot plate with a magnetic stir rod. This approach is thought to improve preservation of tissue morphology as well as efficient retrieval of some antigens. Glycerin has the advantage of having a very high boiling point (290°C) and being nontoxic, stable, and reusable. The stir bar maintains a constant and uniform temperature throughout the antigen retrieval fluid, prevents hot or cold spots, and thus facilitates reliable and consistent results. This method can also be used in a conventional hot air oven. Many slides in metal slide racks can be processed simultaneously in this oven. Glycerin solution can also be used for antigen retrieval in Coplin jars in a microwave oven; an empty space must be kept between the slides. The glycerin method has been used for retrieving a number of antigen types, including estrogen receptor (Beebe, 1999). It is especially useful for very small, fragile biopsies such as prostate needle biopsies and bowel biopsies. Pure glycerin fails to bring about antigen retrieval, which means that water and heat are required to cleave the formaldehyde molecule from the proteins, break down the methylene bridges, or rehydrate the proteins. The exact role of glycerin in the antigen retrieval mechanism is not known. Further testing of the usefulness of this procedure is awaited.

*Contents: chromium potassium sulfate dodecahydrate, sodium dodecyl sulfate, dextran sulfate, formamide, phosphate salt, magnesium sulfate, pepsin, polyethylene glycol, and Triton. It has a low toxicity and is irritating to the eyes and skin. It is a colorless, nonviscous liquid.

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Procedure

The antigen retrieval fluid consists of a mixture of 100 ml of 90% glycerin and 10 ml of Dako’s citrate buffer. The slide rack is placed in a bowl of an appropriate size and shape, so that the stir bar rotates freely under the slide rack. A sufficient volume of antigen retrieval fluid is transferred to the bowl ~ 1 cm above the level of the slides. The hot plate is turned on and adjusted until the temperature of the fluid reaches 100–120°C; the duration of heating varies between 5 and 20 min. For estrogen and progesterone retrieval the duration of heating (120°C) is ~7 min with a 15-min cool-down period before the slides are transferred to distilled water for further processing. Alternatively, such high temperatures in the bowl can be achieved by placing it in a microwave oven, then removing and placing it on the hot plate. This is followed by adding the stir bar and the slide rack to the bowl. It takes 1 min for 100 ml of the glycerin solution to attain a temperature of 125°C in a 600 W microwave oven on high. If more than 100 ml of solution is to be heated to the same temperature, for every additional 100 ml an additional 1 min is required.

pH of Antigen Retrieval Fluids
In addition to heating, retrieval fluid pH plays a key role in achieving optimal antigen retrieval. It is thought that the pH is more important than the composition of the retrieval fluid. This is supported by the demonstration that optimal staining of antigen SMI-32 was achieved at pH 2.5 and 2-hr microwave heating at 90°C, whereas staining of antigen MAP2 was best obtained at pH 4.5 and 10-min full-power heating; in both cases 0.05 M citrate buffer was used (Evers and Uylings, 1994a). Therefore, in optimizing the antigen retrieval protocol, pH is a priority. Note that there is no universally optimal pH for a retrieval fluid. The retrieval of each type of antigen requires a specific fluid pH, although exceptions occur with antigens that can be retrieved at a wide range of pH levels. For example, AEI and NSE (cytoplasmic antigens), PCNA (nuclear antigen), and L26 and EMA (cell surface antigens), can be retrieved at pH levels of 1.0–10.0 (Shi et al., 1995a). On the other hand, some antigens (e.g., estrogen receptor) display a marked decrease in immunostaining at pH 3–6, while still other antigens, such as cytoplasmic HMB45, show weak or negative staining at pH 1-2 but excellent results in the high pH range (Shi et al., 1995a). Some antigens are retrieved only at a low pH. These types are exemplified by thrombospondin and SMI-32 (neurofilament protein), which require pH levels of 1–2 and 2.5, respectively (Grossfeld et al., 1996; Evers and Uylings, 1994a). Note, however, that pH levels lower than 3.0 can severely damage tissue morphology, especially with intense heating. Low pH levels can also alter the localization of some cytoplasmic antigens, resulting in false-positive staining of the nucleus. For example, an antibody (UCHL1) to T cell antigen is effective at pH 6.0 but results in the staining of every nucleus at pH 2.0 (personal communication, H. Y. Lan). Generally Tris-HCl buffer produces better results at higher pH levels (e.g., pH 10.0) than do some other buffers. On the other hand, sodium citrate buffer increases the intensity and extent of immunostaining of a wide variety of tissue antigens at pH 6.0. EDTA-NaOH (1 mM) at pH 8.0 also yields satisfying results. Although relatively high pH solutions, such

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as sodium citrate or Tris-HCl, are suitable for most antigens, low pH solutions are preferred for nuclear antigens (Taylor et al., 1996a). However, solutions of low pH generally tend to cause weak focal background staining and damage to some epitopes. A test battery based on three pH values (low, middle, and high) should be carried out to establish an optimal protocol, including pH, for immunostaining previously unexamined antigens (page 104).

Ionic Strength of Antigen Retrieval Fluids
The ionic strength of the fluid in which tissues are suspended during fixation with formaldehyde, unlike retrieval fluid concentration, does influence antibody access to intracellular antigens such as proliferation cell nuclear antigen (PCNA), nuclear protein (Ki-67) detected by MIB-1 antibody, and nuclear antigen p120. Ionic bonds are known to be responsible for a major portion of protein-protein interactions, and their breakage causes dissociation of the interacting proteins, resulting in increased detectability of the antigen. Such breakage occurs with increased salt (NaCl) concentrations. It has been shown that the immunofluorescence of antigens such as PCNA is increased when the cells are fixed in the presence of increased salt concentrations (Bruno et al., 1992). The increase is greater for cells in the phase of the cell cycle than for cells in S or phase. High salt concentrations loosen the proteins, which are then stabilized with formaldehyde. In other words, increased ionic strength weakens intra- and intermolecular ionic interactions during the process of crosslinking with formaldehyde. Using the optimal ionic strength of the solution, which must be customized for a given antigen, will facilitate the accessibility of the antibody to the epitope.

ANTIBODY PENETRATION
The fundamental question in the phenomenon of antigen retrieval is whether it is due to enhanced penetration of antibodies into the tissue or to reversal of protein conformational changes induced by fixation, or both. The evidence favors both explanations. All the treatments (microwave, autoclave, and conventional heating, enzyme digestion, ultrasound application, and detergent treatment) used for antigen retrieval break down protein crosslinkages, facilitating antibody access to the antigen. The achievement of increased immunostaining after using cell permeabilization methods testifies to the role of antibody penetration into the tissue. The above treatments also restore the original conformation of the protein molecule, resulting in enhanced interaction between the antigen and the antibody. Antibody molecules are relatively large. The speed and extent of antibody penetration into the tissue and the degree of fixation with formaldehyde are inversely related. In other words, the stronger the fixation (protein crosslinking), the slower the antibody penetration. The reason for this relationship is that extensive crosslinking results in the formation of a compact protein network which impedes antibody penetration. Such an impediment can be caused by the cell membranes as well as by the cytoplasmic matrix, both of which contain proteins. It is therefore apparent that strong, extensive protein crosslinking should be avoided before incubation with antibodies. This is the reason for preferring

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formaldehyde over glutaraldehyde, since the latter forms strong protein crosslinkages. It is also well established that tissue specimens that have been fixed for a long time (weeks or months) require more vigorous treatments of sections for antibodies to penetrate and have access to the antigens. It is interesting to note that an antibody against a specific epitope less sensitive to aldehyde fixation can be obtained by immunizing the mice with an antigen in which the aldehyde-sensitive epitope has been blocked or altered. To increase the penetration of antibodies into thin resin sections of fixed tissues, simultaneous heating of sections and antibodies has been attempted. This treatment is thought to increase the labeling of certain antigens, whereas that of some other antigens remains unaffected. It has been demonstrated that such a treatment enhanced labeling density by the antiamylase antibodies, whereas labeling with anti-DAMP antibodies remained unchanged (Chicoine and Webster, 1998). Further developments of this protocol are awaited.

ANTIBODY DILUTION
Not only the type (e.g., the cell clone) and the source of availability of an antibody but also its dilution are important in fully utilizing the effectiveness of an antibody as a powerful tool to detect antigens. The optimal antibody concentration for antigen varies, depending on whether the tissue used is aldehyde-fixed or frozen; generally higher antibody concentrations are required for sections of aldehyde-fixed tissues (Fig. 4.1). Also, different forms of an antigen require different concentrations of the antibody for their maximal detection. This is exemplified by the PC-10 primary antibody, which identifies PCNA antigen at a dilution of 1:1000 in epithelial cells in normal colon tissue, whereas a dilution of 1:400 is required to localize these proliferating cells in adenomatous polyps (Holt et al., 1997). In contrast, some types of antigens (e.g., Ki-67) can be optimally detected in various tissue types at the MIB-1 dilution of 1:50, using the microwave heating antigen retrieval method. However, in a few studies MIB-1 dilutions of 1:20 to 1:100 have been used. There are many pitfalls, including false-positive and false-negative staining, to using antibody concentrations of higher or lower than optimal concentrations. Labeling specificity partly depends on the antibody dilution, while background staining critically depends on its dilution. Also, the esthetic appeal of the images produced by immunohistochemistry diminishes with suboptimal dilutions of antibodies. Therefore, to avoid unwanted staining, pay careful attention to the optimal working dilution of an antibody, especially of a polyclonal antibody, and to washing procedures. Also note that antigen retrieval treatments allow the use of increased dilutions of the antibodies. For example, for sections of formalin-fixed and paraffin-embedded tissues, the optimal dilution of PC-10 antibody without heat pretreatment is 1:10 compared with 1:600 after microwave heating (Haerslev and Jacobsen, 1994). A fairly high concentration of the primary antibody is necessary to follow saturation kinetics. However, the majority of these antibodies exhibits a bell-shaped concentrationbinding curve, with the binding increasing up to a specific antibody concentration and then decreasing. Such a bell-shaped curve is due to unstable binding of the antibody to the antigen under very high antibody concentrations. Effects of a high antibody concentration can be examined with the method of Raivich et al. (1993). In practice, however, one rarely

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aims for or achieves saturation kinetics in routine immunohistochemistry. In most cases we aim for and achieve adequate and reproducible staining. High concentrations of primary antibodies can increase nonspecific binding and also compromise antigen-specific immunostaining. Furthermore, antibodies may aggregate at high concentrations, which limits their penetration. Electrical charges on aggregated antibodies may hinder their penetration among similarly charged cell molecules; therefore, antibodies should be used at concentrations at or slightly below antigen-specific concentrations. Note that similar antibodies obtained from different sources may not yield the same intensities of immunostaining. Some evidence indicates increased labeling efficiency of certain diluted antibodies (e.g., antiamylase antibodies and anti-MHC class II antibodies) when they are exposed to microwave heating prior to their application (Chicoine and Webster, 1998). However, such

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increased labeling may be accompanied by enhanced background staining. Furthermore, such a treatment reduces labeling density of some other antibodies such as anti-DNP antibodies. The duration of heating of antibodies is critical to obtain optimal labeling. The optimal heating duration varies with the antibody and the fixation parameters used to stabilize the cellular components. The mechanism(s) responsible for increased or decreased labeling efficiency of different antibodies as a result of their heating is not known.

Diluent Buffer for Primary Antibodies
Both the pH and osmolarity of the diluent buffer (especially the type of solute) affect the affinity of monoclonal antibodies for the antigen. It is known that electrolytes exert a profound effect, not only on the structural relations of protein molecules, but also on the reactivity of proteins (Hayat, 2000a). The reactivity of both monoclonal and polyclonal antibodies with antigens is affected by the type of antibody diluent used. This is true whether or not a heat-induced antigen retrieval is used. Optimal pH increases the sensitivity (staining intensity) as well as the specificity of immunostaining. Acceptable shelf-life of antibodies can also be achieved at optimal pH and dilution in the presence of stabilizing protein (Boenisch, 1999). Unfavorable pH diminishes immunoreactivity because it reduces antibody affinity for the antigen. The role of pH in the interaction between the antibody and the antigen in immunohistological processing is explained below. Antibodies are attracted to the epitopes of most glycoproteins and polypeptides initially through electrostatic charges and subsequently through van der Waals and hydrophobic interactions (Boenisch, 1999). In immune reactions, the isoelectric point (pI) of both antigens and antibodies is therefore of importance. The pI of polyclonal IgGs ranges from 6.0–9.5. Monoclonal antibodies of at least this class possess an equally wide range of individual pI values. If the pH of the diluent and/or solute is used in the same range, the result will be changes in both electrostatic charge and conformation of at least some monoclonal antibodies and possibly of some reactive epitopes. Antibody configuration controls spatial complementarity. All these changes contribute to variable attraction between the antibody and the antigen. As stated above, the pH of the diluent affects the electrostatic charge of monoclonal antibodies and thus the interactions between the antibody and the reactive epitope. Consequently, the optimal operational pH of the monoclonal antibody is determined by the electrostatic charge of the paratope and that of the epitope. Most effective initial attraction between the paratope and the epitope occurs at the pI intermediate to the antigen and that of the antibody. For most antibodies, but not all, this pH is mildly acidic (6.0). An increasingly higher pH of the diluent will decrease the net positive charge of most monoclonal antibodies, resulting in their reduced attraction to negatively charged target epitopes. Higher pH values will also increase the hydrophobicity of antibodies, lessening the interaction between the antigen and the antibody because of the decreased penetration by the latter. It is suggested that new monoclonal antibodies be tested in several dilutions higher than those recommended by the vendor, using 0.05 M Tris buffer (pH 6.0 and 8.6) (Boenisch, 1999). The highest dilution and the pH at which maximal staining occurs should be determined for routine use of the new antibody. This approach frequently allows for the use of antibody dilutions much higher than those recommended by the supplier.

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Note that the use of higher concentrations of monoclonal antibodies does not improve weak staining in immunohistochemistry because of paucity of or masked antigens. Although PBS is commonly used as the antibody diluent, this solution has certain disadvantages. Sodium ions in PBS tend to shield negatively charged epitopes, thereby diminishing the attraction of positively charged reactive sites on the antibody, especially at an alkaline pH. Phosphate ions, on the other hand, promote hydrophobicity. Therefore, the use of PBS and phosphate buffer for diluting the antibodies is not desirable. Accumulated evidence indicates that the most suitable diluent for both monoclonal and polyclonal antibodies is 0.05–0.1 M Tris buffer (pH 6.0) (Boenisch, 2001). The advantage of this pH becomes clear when considering that most antigens and monoclonal antibodies possess opposite surface charges at pH 6.0. Thus, to achieve optimal immunoreactivity, the pH of the environment should be intermediate between the pH of the antigen and that of the monoclonal antibody. Note that any change in the composition and pH of the diluent affects the performance of both the antibody and the antigen. In addition to the pH, the osmolarity of the buffer used to dilute the primary antibody tends to influence the immunoreactivity of monoclonal antibodies. The changes in the molarity of Tris buffer used for diluting monoclonal antibodies are expected to result in changes in the immunoreactivity of antibodies. It has been reported that the higher the concentration of cations (e.g., ) in the buffer or the higher the pH in their presence, the less the immunoreactivity of the monoclonal antibodies (Boenisch, 1999). However, polyclonal antibodies may not show such an adverse effect.

STORAGE OF PARAFFIN-EMBEDDED TISSUES
Antigenicity is preserved much better in paraffin-embedded tissue blocks during storage than on the paraffin sections. However, an agreement is lacking regarding the loss of antigenicity due to storage of formalin-fixed and paraffin-embedded tissues. Even similar tissues processed in a similar fashion and stored for the same period of time in different laboratories may show differences in the degree of immunoreactivity. Also, the use of the same antibody when used in different tissues stored for the same duration may yield different degrees of immunohistochemical staining. This predicament is exemplified by the androgen receptor. Androgen receptor activity has been reported to be preserved in the sections of formalin-fixed, paraffin-embedded human archival benign prostate tissue that was stored for up to 16 years (Janssen et al., 1994). In this study monoclonal antibody F39.4 was used; it was raised against a synthetic peptide (SP61) corresponding to the human androgen receptor amino acid sequence 301–320 of the N-terminal domain. In contrast, it has been demonstrated that a significant and persistent decrease in the androgen receptor immunoreactivity occurred in prostatic adenocarcinomas when they were stored for 2 years (Dash et al., 1998). The antibody used was F39.4.1 (BioGenex, San Ramon, CA). Such an immunoreactivity decreased to near zero after 4 years. This decrease begins slowly, followed by more rapid decline, and finally again slows down. In this study, antigen retrieval with microwave heating did not negate the adverse effects of the storage of tissue blocks. Methyl green was used for identifying the tissue background and highlighting the nuclei. Image acquisition and analysis were performed with a CAS 200 image

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analyzer (Becton Dickenson Cell Analysis Systems, Mountain View, CA). Antigen retrieval in both of these studies was carried out using microwave heating. If the preservation of androgen receptor antigenicity is a problem in a formalin-fixed archived tissue, one way to circumvent this problem is to use archived fresh-frozen tissues. On the basis of their study, Dash et al. (1998) have suggested that the use of this antibody for retrospective studies does not correlate androgen receptor status with prognosis or therapeutic response. A decrease in immunoreactivity of p53 antigen in stored paraffin sections is discussed on page 85.

STORAGE OF TISSUE SLIDES
Sections of formalin-fixed and paraffin-embedded tissues are commonly used to detect antigens of diagnostic, therapeutic, or prognostic importance in patients with many types of cancers. Therefore, the need for accuracy in the detection of antigen is obvious. Although sections of these tissues are mounted on glass slides usually 1 or 2 days before staining, in some cases paraffin blocks are no longer available. Consequently, immunohistochemistry must be performed on unstained slides that have been prepared some time ago and stored. Because antigen alterations occur on unstained, stored paraffin sections, factors responsible for the alterations need to be understood in order to increase the reliability and quality of immunohistochemical studies (Hayat, 2000a). Most of the technical factors that positively or negatively affect antigen detections are discussed elsewhere in this volume. The following discussion is limited to the clarification of factors influencing the detection of antigens on the stored paraffin sections. Generally, prolonged storage of sections at room temperature results in decreased immunostaining, and thus false-negative staining, which may lead to diagnostic errors and inaccurate prognostic information (Fig. 4.2/Plate 1B-E). In general, antigens that do not require antigen retrieval assistance are less adversely affected during storage than those requiring antigen retrieval with microwave heating or enzyme digestion. Membranous antigens seem to be more adversely influenced by storage of sections than are those located in the cytosol and the nucleus. It is emphasized that antigens affected during storage of sections show decreasing staining with increasing storage temperatures because the stability of most antigens remains intact during storage at 4°C. Decreased immunoreactivity caused by section storage can be compensated for in most cases by using the optimal antigen retrieval method. Different adhesives, such as gelatin and poly-L-lysine, used to ensure section adhesion to the slide do not influence antigen preservation during storage (van den Broek and van de Vijver, 2000). Admittedly, the effect of storage of paraffin-embedded tissue sections on the extent and intensity of immunostaining is controversial. A number of contradictory reports have been published, especially regarding the effects of the type of fixative, duration of fixation with formalin and other fixatives, and temperature of storage (Leong and Gilham, 1989b; Miettinen, 1989; Malmström et al., 1992; Hendricks and Wilkinson, 1994; Bromley et al., 1994; Prioleau and Schnitt, 1995; Kato et al., 1995; Jacobs et al., 1996; McDermott et al., 1997; Shin et al., 1997; Songun et al., 1997; Bertheau et al., 1998; Grabau et al., 1998; Dwork et al., 1998). Recently, excellent, detailed studies of this problem have been carried

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out by Wester et al. (2000) and van den Broek and van de Vijver (2000). These studies and my personal experience are reviewed below. Only a few studies report that prolonged storage of sections does not adversely affect antigen detectability. For example, according to Williams et al. (1997), long-term (6 months at room temperature) storage of sections of tonsil tissue had no effect on the reactivity of the five antibodies tested. In contrast, a vast number of other studies demonstrate decreased staining of stored sections, especially when they are stored at room temperature. It has been demonstrated, for example, that antigens such as p53 and Ki-67 (lung and breast carcinoma) show lower staining after storage for 3 years at room temperature than sections stored for the same period of time at 4°C or –80°C (Grabau et al., 1998). Nuclear estrogen receptor in breast carcinoma also shows higher reactivity when deparaffinized sections are stored for up to 4 weeks in 10% sucrose in PBS at 4°C than that shown by sections stored at room temperature for the same duration (Bromley et al., 1994). This increased staining could be the effect of cold temperature and PBS in which the sucrose is dissolved. It is also known that dissolved salt solutions unmask epitopes in formalin-fixed and paraffin-embedded tissues. It has also been demonstrated that the staining of p53 in mammalian ductal carcinoma decreased after slides were stored for 2 months at room temperature, but the antigen loss

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was significantly less when slides were stored at 4°C (Jacobs et al., 1996). A gradual loss of staining of Ki-67 was reported in the colon tissue when the slides were stored for 9–21 days at room temperature before staining, although a delay of 5 days did not diminish the staining (Holt et al., 1997). Maintaining cut sections refrigerated and protected from light failed to prevent such a loss of MIB-1 immunoreactivity with time. On the other hand, when sections were stored for as long as 1 month at room temperature before staining of PCNA antigen in the same tissue, no adverse effect was observed on the immunoreactivity of PC10 antibody with this antigen. According to Shin et al. (1997), p53 immunoreactivity was not decreased with storage of slides for as long as 25–48 months at room temperature, provided staining intensity is not the only objective of the study. The percentage of positivity of microwave-enhanced immunoreactivity of p53 stored at room temperature and fresh paraffin sections was not statistically significant. Nevertheless, the staining intensity of heated, stored sections was stronger than that in nonheated, freshly cut sections. This study was carried out using tissue blocks of head and neck squamous cell carcinomas stored for 4–15 years and lung carcinomas stored for 14–25 years. Zinc sulfate (1%) was used as the antigen retrieval fluid and was heated for 3 min in the microwave oven. Similarly, the immunostaining of p53 antigen in sections of colorectal carcinoma stored for 6–14 months at room temperature was excellent after microwave heating (Kato et al., 1995). The aforementioned disagreement is due to the study of p53 antigen in different tissues and/or differences in the details of the methodologies used in different laboratories. There are many reasons for the lack of consensus on this highly complex phenomenon, and they are discussed below. Various studies mentioned above were conducted using different parameters of antigen retrieval methods, including antigen retrieval fluids, pH, heat source, temperature, and duration of treatments for detecting different antigens. The type of fixation and duration of fixation also varied in these studies. Other variants were the type of epitope and antibody and source of antibodies used. The degree of immunostaining of stored paraffin sections may differ, depending on whether monoclonal or polyclonal antibodies are employed. Polyclonal antibodies have affinity for several types of epitopes, resulting in positive staining, which may be nonspecific. An antigen retrieval method unmasks more than one type of epitope on the same section, whether stored or not, and such epitopes have access to the polyclonal antibody. However, a recent study indicates that polyclonal antisera show only slightly better staining than that obtained with monoclonal antibodies (van den Broek and van de Vijver, 2000). It is also possible that loss of immunostaining in stored sections is epitope related instead of related to the antigen as a whole (Henson, 1996). In addition, tissue heterogeneity at different levels was not considered. The quantity and quality of antigen reactivity varies from one tissue block to another. In some of these studies the automated immunostainer was used, whereas in others manual staining was carried out. The automated stainers reduce human procedural errors, and their controlled environment increases the speed and timeliness of results in high-volume laboratories, although their high cost might be prohibitive for small laboratories. Another factor that may affect results is that a collection of stored sections may be heterogenous regarding duration of storage because of the successive addition of new sections. Subjective evaluation or inadequate quantification of the extent and intensity of staining was the common

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denominator in most of these studies. These are the main reasons for the contradictory results reviewed above. Attempts have been made to protect sections from exposure to oxygen during storage by coating them with paraffin (Jacobs et al., 1996; Rittman, 2000). This is accomplished by heating the slide to ~60°C and placing a few drops of molten paraffin on it; a second heated slide is gently drawn across the surface of the first slide to form a thin protective paraffin layer on the sections. In our experience, coating the surface of paraffin sections mounted onto slides with paraffin does not significantly reduce antigen loss after their storage for several weeks or months at room temperature or at 4°C. Xylene-based spray glue has also been used for protecting the stored sections but without success (Wester et al., 2000). Different antigens are affected differently by the storage of sections of even the same formalin-fixed, paraffin-embedded tissue. In other words, the degree of the antigenicity loss due to storage of the unstained slides differs depending on the type and location of the antigen. For example, nuclear steroid receptors tend to be comparatively more sensitive to storage (aging). Comparative studies, using a panel of eight antibodies against Ki-67, prostatic-specific antigen, androgen receptor, epidermal growth factor receptor, and prostatic acid phosphatase, demonstrated that nuclear androgen receptor showed a higher decrease of antigenicity in stored, unstained sections compared with that exhibited by other antigens (Olapade-Olaopa et al., 2001). The loss of antigens due to storage of paraffin sections is not a serious problem in many clinical immunohistochemistry laboratories that perform immunostaining within hours or days after paraffin sections have been cut. However, antigen loss may become a problem when slides are stored for months at room temperature as positive controls. Such storage is encountered in some research laboratories where unstained paraffin sections are archived for future use. In any case it is recommended that sections be stained rapidly after they have been cut from the formalin-fixed, paraffin-embedded tissues. It is likely that prolonged storage of sections at room temperature strengthens protein crosslinks, which become less reversible, resulting in diminished antigen retrieval. If immunostaining needs to be postponed, tissue specimens should be stored in paraffin blocks rather than as paraffin sections because antigenicity is better preserved in the former state. The results of a comprehensive study on the effects of fixation, temperature and duration of section storage, and antigen retrieval on the immunostaining of p53 antigen are shown in Fig. 4.3 (Plate 2). Color-based image analysis was used to quantify the extent and intensity of staining. In conclusion, the storage of paraffin sections decreases, to a varying degree, immunoreactivity for most, but not all, antigens. The maximal decrease in immunoreactivity, at least of p53 and Ki-67 antigens, occurs during the first 2 weeks of storage (Wester et al., 2000). The decrease in immunoreactivity is generally inversely related to an increase in storage temperature. Both the extent and intensity of staining tend to be negatively influenced by storing the sections. The decreased immunoreactivity as a result of section storage can be compensated for in most cases by using optimal antigen retrieval procedure. Although longer durations of fixation are accompanied by increased masking of antigens during section storage, this relationship is neither universal nor linear. If paraffin sections must be stored, they should be stored at –20°C, irrespective of the duration of storage.

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SIGNAL AMPLIFICATION
Most antigens are not destroyed during fixation with formaldehyde, but are reversibly masked. Methods to unmask them are presented in this volume. However, some antigens are difficult to visualize adequately with routine immunohistological techniques and therefore require signal amplification with an acceptable signal-to-noise ratio. An amplification of immunostaining intensity is especially useful when monoclonal antibodies are used because they bind only a single epitope. Small amounts of antigens in tissue sections can be detected specifically by using signal amplification. Techniques used for increasing the sensitivity or signal amplification are summarized below. A number of strategies have been employed for improving immunohistochemical signals. The immunofluorescence antibody method was developed for specific identification of cells based on their antigen makeup (Coons et al., 1941). Its use is limited because of the need for fresh-frozen sections and inadequate preservation of cell morphology. Also, the fixed ratio of fluorescein to the antibody does not allow amplification of the signal. The peroxidaselabeled antibody method is more compatible with the basic substrates of surgical pathology specimens fixed with formalin and embedded in paraffin (Nakane, 1968). This immunoperoxidase protocol can be amplified by increasing the duration of development. The original immunoenzyme bridge method using enzyme-specific antibody (Mason et al., 1969) has been superseded by an improved technique using a soluble peroxidase antiperoxidase complex (PAP) (Sternberger et al., 1970). These complexes are formed from three peroxidase molecules and two antiperoxidase antibodies and are used as a third layer in the staining method. They are bound to the unconjugated primary antibody (e.g., rabbit antihuman IgG) by a second layer of bridging antibody (e.g., swine antirabbit immunoglobulin), which is applied in excess so that one of its two identical binding sites binds to the primary antibody and the other to the (rabbit) PAP complex. The PAP method is more sensitive than indirect methods using fluorescein or peroxidase-conjugated antisera. Alkaline phosphatase antibodies raised in the mouse can, by the same principle, be used to form alkaline phosphatase anti-alkaline phosphatase (APAAP) complexes. These have uses and advantages similar to those of the PAP complexes. The avidin-biotin methods rely on the marked affinity of the glycoprotein avidin for biotin. Avidin is composed of four subunits which form a tertiary structure possessing four biotin-binding hydrophobic pockets. The oligosaccharide residues in avidin give it some affinity for the tissue components, especially some lectinlike proteins, and result in nonspecific binding. A similar molecule, streptavidin, has some advantages over avidin, as the former lacks oligosaccharide residues and possesses a neutral isoelectric point. The low-molecular-weight vitamin biotin is easily conjugated to antibodies and enzyme markers. Up to 150 biotin molecules can be attached to one antibody molecule, and the strong affinity of the biotin for the glycoprotein avidin allows its use as complexing secondary reagents. Biotin labeling of the primary (direct) or secondary (indirect) antibody can be used in the avidin-biotin methods. In the labeled avidin method the tracer is attached directly to the avidin molecule. In the avidin-biotin bridge method a biotinylated enzyme such as peroxidase is allowed to bind after attachment of avidin to the biotinlabeled antibody. In the avidin-biotin (ABC) method a complex of avidin and biotinylated tracer containing the free avidin binding sites is applied to the biotinylated antibody. As a high

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number of biotin molecules can be attached to a single antibody, a high tracer-to-antibody ratio can be achieved. This property yields high sensitivity and allows the use of an increased dilution of the primary antibody. The most recently developed and refined signal amplification method is termed catalyzed reporter deposition (CARD), which is discussed below. The historical development of signal amplification methods has been presented by Elias (1999).

Tyramine Amplification Method
The tyramine amplification method is based on the characteristic ability of tyramine to become chemically adhesive following oxidation/radicalization (Gross and Sizer, 1959). Oxidation of the tyramine generates a brown pigment that contains dityramine and more extensively oxidized and polymerized derivatives. Bobrow et al. (1989, 1991, 1992) used tyramine for enhancing ELISA, and Adams (1992) adapted it for immunohistochemistry. Subsequently, Mayer and Bendayan (1997) extended the application of the tyramine signal amplification to immunoelectron microscopy (Fig. 4.4). Van Gijlswijk et al. (1997) used green, red, and blue fluorescent tyramides in immunohistochemistry, immunocytochemistry, and fluorescence in situ hybridization. Other applications of tyramine include Western blotting (Wigle et al., 1993) and in situ hybridization (Kerstens et al., 1995). Tyramine amplification is an important development in advancing the efficiency of immunohistochemistry, and its further applications are expected. The catalyzed reporter deposition (CARD) amplification signal method was initially described by Bobrow et al. (1989). It is based on the deposition of biotinylated tyramine at the location of the probe catalyzed by horseradish peroxidase (HRP). It has been established that the highly reactive intermediates formed during the HRP-tyramide reaction will bind to tyrosine-rich moieties of proteins present in the vicinity of the HRP binding sites. The binding of tyramine to proteins at the site of HRP occurs via the production of free radicals by the oxygen liberated by HRP. In other words, HRP reacts with and the phenolic moieties of tyramine to produce a quinonelike structure bearing a radical on the C2 group. Because this reaction is very short lived, deposition occurs only in the location at or in immediate proximity to where it is generated. The biotin conjugated to the bound tyramine is subsequently used for the attachment of avidin, which is conjugated to HRP. This HRP is then used to catalyze the brown color reaction. This method allows highresolution detection of primary antisera because the tyramide complex precipitates only at the site of reaction. Toda et al. (1999) have compared the immunostaining using conventional avidin-biotin complex (ABC) with tyramide signal amplification-avidin-biotin complex (TSA-ABC); relatively distinct staining was apparent in the latter technique. As the TSA-ABC protocol dramatically improves the signal intensity by the peroxidasecatalyzed deposition of biotinylated tyramide, blocking of endogenous peroxidase is required. To ensure quenching of the residual peroxidase, the use of a higher concentration and duration of treatment with is recommended. The amplification power of TSA-ABC can be enhanced by using several subsequent cycles of incubation or by extending the duration of incubation, for example, up to 30 min at 37°C. Longer incubations may result in background noise (nonspecific staining). Also, the number of cycles possible before the background noise level becomes unacceptable is

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limited to two or three. It should be noted that because the tyramide deposition reaction is rapid, small differences in amplification duration may lead to variations in the final signal intensities. Furthermore, because this reaction amplifies both specific and nonspecific immunohistochemical signals, it is essential that appropriate positive and negative controls be used to achieve correct interpretation of staining. Also, because TSA-ABC tends to enhance the background noise along with the signal, the procedure must be optimized to ensure low nonspecific binding. All tyramide conjugates yield approximately the same

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results, indicating that signal amplification is independent of the tyramide conjugate used (Speel et al., 1998). Numerous biotin conjugated tyramides can be detected with avidinconjugate (Totos et al., 1997). If biotin reaction fails, the primary reason is the age of the biotin solution. Because the shelf-life of newly synthesized biotin is not known, one should at least be aware of the expiration date of the reagent. Different authors and commercial suppliers have assigned different names to signal amplification using tyramine. For example, tyramine signal amplification (TSA) system and the catalyzed signal amplification (CSA) system are commercially available from DuPont NEN Life Science Products, Boston, MA, and DAKO Corporation, Carpinteria, CA, respectively. In addition, the terms CARD (catalyzed reporter deposition) (Bobrow et al., 1989), TA (tyramide amplification) (Shindler and Roth, 1996), and ImmunoMax (Merz et al., 1995) have been used for the tyramine amplification technique. The use of different names for almost identical procedures has resulted in confusion. To standardize the terminology, the neutral abbreviation, tyramide amplification technique (TAT) should be accepted (Von Wasielewski et al., 1997). One of the variations of the tyramide amplification technique is termed ImmunoMax (Merz et al., 1995). In this approach the biotinylated tyramine enhancement is combined with an antigen retrieval method such as microwave heating, enzyme (proteinase K) digestion, or exposure to a detergent (guanidine hydrochloride). This method is effective in detecting some previously unreactive, inadequately reactive, or partly demasked antigens in the formaldehyde-fixed and paraffin-embedded tissues. It has been claimed that this technique allows as much as 10,000-fold dilution of the primary antibody and 100 to 1,000-fold increase in sensitivity compared to those used with the conventional ABC method (Merz et al., 1995). However, the sensitivity increase in the range of 5- to 50-fold is more feasible (Speel et al., 1999).
Preparation of Biotinylated Tyramine

One hundred milligrams of sulfosuccinimidyl-6- (biotinimide) hexanoate (NHS-LCbiotin) (Pierce, Rockford, IL) is dissolved in 40 ml of 50 mM borate buffer (pH 8.0). To this solution is added 30 mg of tyramine hydrochloride (Sigma Chemical Company, St. Louis, MO). The solution is stirred overnight at room temperature and filtered ( filter). The final biotinylated tyramine concentration is Before application, the solution is diluted 1:160 in Tris-HCl buffer (pH 7.6) containing 0.03%

Rolling Circle Amplification
Although immunohistochemistry is a versatile and powerful tool for various molecular and cellular analyses, especially for antigen detection, it has a few limitations, such as lack of standardization and difficulty in visualization of antigens present at low concentrations. In numerous instances important biological markers for cancer, infectious disease, and biochemical processes are present at too low a concentration in tissues or body fluids to be detected by conventional methods. The difficulty of detection of low concentrations of antigens can be minimized by antigen retrieval using heating or enzymatic digestion.

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This problem can also be lessened by using stronger fluorochromes and chemiluminescent substrates for use in ELISAs, immunofluorescence-based staining, and immunoblotting. Detection of low concentrations of antigens can also be achieved by increasing the signal without raising the level of nonspecific background staining. Signal amplification, for example, can be achieved by successive steps of enzymatic reactions. Biotinyl tyramide is commonly used to increase the signal of low abundance targets that are otherwise undetectable by conventional techniques. However, tyramide-based amplification may increase background noise because of multiple steps of signal amplification (discussed in this chapter). Therefore, molecular tissue pathology requires techniques of greater sensitivity and specificity. One of such techniques to refine the examination of cell components is rolling circle amplification (RCA) discussed below (Lizardi et al., 1998). Rolling circle amplification is essentially a surface-anchored DNA replication that can be used to visualize single molecular recognition events. It is an isothermal nucleic acid amplification protocol that differs in several aspects from the polymerase chain reaction (PCR) and other nucleic acid amplification methods. The RCA can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions. It has sufficient sensitivity to detect individual oligonucleotide hybridization events and single antigen-antibody complexes (Schweitzer et al., 2000). The linear mode of RCA can generate signal amplification during a brief enzymatic reaction. Another advantage of linear RCA is that the product of amplification remains connected to the target molecule. Signal amplification by RCA can be coupled to nucleic acid hybridization and multicolor fluorescence imaging to detect single nucleotide changes in DNA within a cytological context or in single DNA molecules (Zhong et al., 2001). This protocol has been used for visualizing target DNA sequences as small as 50 nts long in peripheral blood lymphocytes or in stretched DNA fibers (Zhong et al., 2001).

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Chapter 5

Problems in Antigen Retrieval

LACK OF IMMUNOSTAINING
The failure of antibodies to immunostain tissues or cells does not necessarily reflect the absence of epitopes. Lack of, or reduced, immunostaining can be attributed to multiple factors. The most common factor is the inability of the antibody to reach and recognize the epitope under the preparatory conditions used, including fixation, dehydration, embedding, deparaffinization, rehydration, and incubation. The inaccessibility of the epitope to the antibody may be due to the formation of large, compact protein complexes as a result of crosslinking by formaldehyde. These complexes create a barrier to antibody penetration. This aspect of immunostaining failure is elaborated upon later. It is also possible that the antigen molecule is folded and thus hides, the epitope, especially from monoclonal antibodies. Apparently, better immunostaining depends on improving antibody access to, and recognition of, the epitope. It is well established that many types of antigen molecules are altered by dehydration solvents and other reagents. Lack of immunostaining may also be due to excessively diluted antibody, to loss of antibody owing to degradation by bacteria or fungi, or to antibody aggregation due to repeated freezing and thawing. Finally, a monoclonal antibody will not recognize an epitope in vivo if the former is raised against a denatured antigen. This is also true for polyclonal antibodies when the recombinantly produced antigen becomes denatured during isolation and purification (Binder et al., 1996). Fixation with aldehydes plays a key role in the two above-mentioned events: antibody access to and recognition of the epitope. Tissues and cell cultures are usually fixed with an aldehyde prior to immunostaining. Fixation has the advantage of anchoring in situ antigens, as aldehydes are powerful protein crosslinking agents. They crosslink proteins and glycoproteins through reversible and irreversible alterations in the molecular conformation of proteins, including antigens (epitopes). If the change in conformation is strongly irreversible, the antibody will have difficulty recognizing the altered epitope, especially aldehyde-sensitive antigens. This problem is especially acute when specimens are fixed with glutaraldehyde. This effect of aldehydes on epitopes and their surrounding proteins is called epitope masking. Briefly, epitope unmasking can be accomplished by weakening or breaking down the protein crosslinking introduced during aldehyde fixation and allowing the epitope to be exposed to the antibody, provided the latter has access to the former. Thus, two simultaneous events (unmasking of epitope and access of antibody to epitope)
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must take place to achieve immunostaining. Detailed methods of unmasking or retrieval of epitopes after fixation with an aldehyde are discussed later. Note that there is no single, ideal pretreatment for retrieving all types of epitopes; the optimal treatment for a given epitope may not be effective for another type of epitope. This is particularly true when considering the pH of the epitope retrieval solution and the duration of pretreatment. These and other pretreatment conditions are explained later. Note also that less than optimal processing conditions for a given epitope may also result in background immunostaining.

BACKGROUND STAINING
Background staining is one of the common problems in immunohistochemistry, and it has a number of causes, which are discussed below. One cause is protein hydrophobicity, which can occur between different protein molecules. Fixation with aldehydes renders proteins more hydrophobic as a result of crosslinking between reactive amino acids. The crosslinkages are both intramolecular and intermolecular (Hayat, 2000a). The extent of hydrophobic crosslinking depends on the duration, temperature, and pH of fixation. Because changes in these factors result in variable hydrophobicity, owing to variable protein crosslinking, once an optimal fixation is determined, it must be maintained and controlled. According to Boenisch (2001), excessive background staining resulting from overfixation with formalin can be remedied by postfixation with Bouin’s or B5 fixative. It should be noted that the greater the proximity of the pH of the antibody diluent and the isoelectric point (pI) of the antibody, the stronger the hydrophobic interaction. In contrast, the lower the ionic strength of the diluent, the weaker the strength of hydrophobic attraction. Hydrophobic interactions can also be reduced by adding a detergent, such as Tween 20, to the antibody diluent. The best approach to significantly reduce background staining due to hydrophobic interaction is to use a blocking protein immediately before or also during the application of the primary antibody. The blocking protein must be of the type that competes effectively with IgG for hydrophobic binding sites in the tissue. Also, the blocking protein should be identical to that present in the secondary link or labeled antibody, but not that in the primary antibody, in order to avoid nonspecific binding of the secondary antibody. To fulfill these requirements, 1% bovine serum albumin (BSA) is added to the primary antibody diluent. Nonfat dry milk or casein can be used in place of BSA. The cross-reactivity of antibodies can also cause background staining. This problem arises when the epitope under study is shared among different proteins in the target tissue. Use of polyclonal antibodies can result in nonspecific cross-reactivity with similar or dissimilar epitopes on different antigens. Because an unabsorbed antiserum tends to increase this problem, it should be subjected to careful affinity absorption. Use of antibodies from hyperimmunized animals will also help. Careful screening of clones in the case of monoclonal antibodies will eliminate this type of background staining. Antibody crossreactivity has been discussed in more detail in Chapter 2. The presence of even small amounts of natural antibodies in the serum may also produce nonspecific staining. These antibodies result from prior environmental antigenic stimulation. In fact, such antibodies may increase in the titer during immunization of the

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animal with adjuvants. Although these antibodies are difficult to remove, their net effect can be almost eliminated by using the antiserum at a sufficiently high dilution or by reducing the duration of incubation. Nonspecific staining may also result from contaminating antibodies produced by the host’s immune system as it reacts to isolated antigens used for immunization (Boenisch, 2001). Isolated antigens are rarely pure. If these antibodies are a problem, the antiserum should be subjected to affinity absorption. Fortunately, such antibodies are present in a very low concentration and may not cause troublesome background staining. Use of hightitered antisera at sufficiently high dilutions would eliminate this problem. Natural and contaminating antibodies do not cause any problem when using monoclonal antibodies. Nonspecific staining can be caused by Fc receptor glycoproteins present on the cell membrane. This problem is more relevant to frozen sections and smears than to tissues fixed with formaldehyde. The problem can be avoided by using fragments instead of whole IgG molecules (Boenisch, 2001). Complement-mediated binding may also cause background staining in frozen sections when whole antisera is used; however, this problem is not very common. Antigen diffusion can cause specific background staining. This problem arises when the target antigen is displaced from its site of synthesis or storage. Delayed fixation and/or incomplete fixation with formaldehyde tend to cause this problem. Optimal fixation with this monaldehyde anchors the antigens at their site of synthesis. Mechanical injury to the tissue or drying of the tissue prior to fixation may result in diffuse background staining. Necrotic areas due to autolysis of the tissue tend to stain with almost all staining reagents. Antigen retrieval with prolonged enzyme digestion often disrupts cell architecture, resulting in the displacement of target antigens from their site of greater density; the net effect is increased background staining. Background staining also results from the presence of endogenous peroxidase in the formalin-fixed tissues. This artifact can be avoided by treating the tissue sections with 3% hydrogen peroxide in water for 4–9 min at room temperature; methanolic hydrogen peroxide is not recommended. Blocking of the endogenous peroxidase activity is especially desirable with cell preparations and frozen sections (Boenisch, 2001). Endogenous biotin, distributed in a wide variety of tissues, may also cause background staining with biotin-based immunohistochemical techniques. This biotin is especially abundant in liver, whereas it is poor in the central nervous system and adipose tissue. Endogenous biotin activity is more abundant in the cytoplasm and cryostat sections but is also present in sections of paraffin-embedded tissues. This problem is largely eliminated by using streptavidin-based methods or by sequential treatment of sections (prior to staining) with 0.01–0.1% avidin followed by 0.001–0.01% biotin for 10–20 min each. The biotin problem is discussed in more detail later in this chapter. Other causes of diffuse background staining include the presence of residual embedding medium and bacterial or yeast contamination in the water bath. The presence of undissolved chromogen granules on occasion may create the problem of nonspecific staining. Excessive counterstaining with reagents, such as hematoxylin and eosin, may compromise specific staining. Finally, a few published reports indicate that antigen retrieval at extremely high temperatures may result in nonspecific staining. Baas et al. (1996), for example, have reported false-positive results at a very high antigen retrieval temperature using monoclonal

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antibody D07 against p53 antigen. They carried out antigen retrieval at 96°C for 30 min in the Target Unmasking Fluid (TUF) containing 35% urea in a microwave oven. This combined treatment is unusually excessive and is not used routinely.

PROBLEM OF ENDOGENOUS BIOTIN
The presence of endogenous biotin is a known potential source of nonspecific staining in immunohistochemical methods based on the avidin-biotin system (Fig. 5.1/Plate 3A and B). Biotin is a water-soluble monocarboxylic acid (a vitamin) of molecular weight 244 Da in living cells. This vitamin functions as a prosthetic group for carboxylase enzymes used in fatty acid biosynthesis and gluconeogenesis. It is widely distributed in many tissue types, including liver, kidney, breast, pancreas, salivary glands, skeletal and cardiac muscles, adipose tissue, and a variety of neoplasms (e.g., salivary gland neoplasm [Lu et al., 2000]). Biotin has been demonstrated immunohistochemically, for example, in human thyroid, parathyroid, adrenal, salivary, mammary, and prostate glands (Green et al., 1992). The presence of cytoplasmic endogenous biotin has also been demonstrated in thyroid papillary carcinoma (Kashima et al., 1997). This vitamin is found both in the cytoplasm and in the mitochondria. The avidin-biotin complex (ABC) method and the streptavidin-biotin (SAB) method are more sensitive than the peroxidase-antiperoxidase (PAP) method for histochemical techniques. The strong noncovalent attraction between biotin and avidin or streptavidin is exploited in many histochemical, immunohistochemical, and in situ hybridization

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procedures discussed in this volume. As an example, avidin-peroxidase and streptavidinperoxidase conjugates and antibiotin antibodies are routinely used for labeling biotinylated antibodies that bind to antigens. In addition, antibiotin antibodies are employed in multistep protocols to enhance the sensitivity of immunohistochemical and in situ hybridization methods (McQuaid and Allan, 1992). However, the avidin-biotin and the streptavidin-biotin detection systems in some cases can lead to false-positive immunostaining. The avidin conjugates employed in these biotinavidin methods bind to the endogenous biotin, thereby resulting in artifactual staining. Such spurious staining has been documented in the above-mentioned tissues as well as in such disparate tissues as gestational endometrium and ovarian lipid cell tumors (Seidman et al., 1995). Another example of false-positive immunostaining is the purported presence of inhibin in hepatocellular carcinomas (McCluggage et al., 1997). However, a recent study has demonstrated that when endogenous biotin is blocked, immunostaining of inhibin in hepatocellular carcinomas and hepatocytes is absent (lezzoni et al., 1999). By blocking endogenous biotin, highly specific staining of endothelium using CD-34 as an antibody without nonspecific tubular staining has been achieved in the kidney tissue (Rodriguez-Soto et al., 1997). The frequency of such erroneous staining is likely to increase as the sensitivity of the protocols for labeling biotin improves (i.e., biotin amplification techniques) (Adams, 1992). It is thought that the problem of endogenous biotin staining is more serious with some antibiotin antibodies than with streptavidin conjugates (Cooper et al., 1997). The problem depends on the affinity/sensitivity of the antibody used. Also, the problem becomes more prevalent when tissues are pretreated with detergents or digestive enzymes for antigen retrieval (Satoh et al., 1992). Moreover, the intensity of this artifact is enhanced by heat-induced antigen retrieval methods. This nonspecific staining is also observed in in situ hybridization with biotinylated probes (Kashima et al., 1997). The presence of this artifact poses a distinct risk of its being interpreted as positive staining, as the artifact can be intense and may be precisely located in the cells of interest with a clean background. It is known that liver and kidney can retain high amounts of retrievable biotin-avidin activity in neoplasms. The need for adequate controls or biotinblocking procedures is obvious when histochemical or immunohistochemical procedures are used. Negative controls facilitate identification of such nonspecific staining. An alternative to the avidin-biotin technology, the EnVision™+System (Dako) detection method, is recommended for universal use in diagnostic and research studies. It is based on enhanced polymer methodology. In comparison with APAAP, PAP, ChemMate™, CSA, LABC, and SABC methods, the En Vision™+System yields optimal detection (Sabattini et al., 1998). Its sensitivity is at least as good as that of Strept ABC techniques, and its use completely eliminates the problem of endogenous biotin. Another more recently introduced method to prevent endogenous biotin staining consists of using a nonbiotin amplification (NBA) detection system (Zymed, San Francisco, CA) (Shi et al., 2000b). This method is as effective as the conventional technique using the Lab-SA kit (Histostain-Plus kit, Zymed) but avoids nonspecific biotin staining. The NBA kit is composed of an FITC-labeled secondary antibody and a horseradish peroxidase– conjugated anti-FITC antibody. Figure 5.2 (Plate 1F and G) shows HER-2 staining in the infiltrating ductal carcinoma cells of breast using the Lab-SA kit or the NBA kit. If either of the two methods mentioned above is not used, the following procedure can be employed to avoid endogenous biotin staining.

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Endogenous biotin-avidin activity can be blocked by treating the sections of formalinfixed and paraffin-embedded tissues with avidin solution alone or followed by biotin solution. These treatments are carried out after heating for antigen retrieval, and they do not interfere with subsequent immunoperoxidase staining. The concentration of the avidin is critical in blocking endogenous biotin. A cost-effective alternative to commercially available avidin, which is rather expensive, is dilute egg white (Miller and Kubier, 1997). Avidin solution can be prepared by mixing two egg whites in 200 ml of distilled water. Skim milk should not be used as a substitute for commercially available biotin (R.T. Miller, personal communication). Use of 0.2% biotin in PBS is recommended. The specificity of this method requires the absence of both avidin-binding sites and peroxidase activity in the tissue sections. Immunoperoxidase staining of endogenous biotin in frozen sections can be eliminated by their treatment with 1% hydrogen peroxide in methanol (Cooper et al., 1997). Such treatment should be applied after application of primary antibody.

Procedure (lezzoni et al., 1999)
Sections ( thick) of formalin-fixed and paraffin-embedded tissues are placed on slides, dried overnight at 37°C, deparaffinized with xylene, and rehydrated with descending concentrations of ethanol. They are treated with 3% hydrogen peroxide in methanol for 5– 10 min and then rinsed with PBS. The slides are immersed in 10 mM citrate buffer (pH 6.0), and heated for 2 min at 100% power, followed by 8 min at 80% power. After being rinsed in PBS, the sections are incubated in an appropriate primary antibody. They are rinsed in PBS and then treated for 4–8 min with egg white avidin solution (2.5 g of egg white avidin in 0.1 mM PBS) (Ventana Medical Systems). Avidin binds to endogenous biotin. Following rinsing in PBS, the sections are treated with free biotin solution (2.5 mg in 0.1 mM PBS) for 4 min to saturate the remaining binding sites of the egg white avidin. The sections are thoroughly rinsed with tromethamine-based buffer or PBS, followed by the following sequential immunostaining protocol: biotinylated secondary antibody,

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avidin-strep+avidin-horseradish peroxidase conjugate, DAB-hydrogen peroxide, copper enhancer, and hematoxylin. The slides are dipped in lithium carbonate, and cover-slipped.

MIRROR IMAGE COMPLEMENTARY ANTIBODIES
The conventional immunodetection system may contribute to poor staining specificity because of unwanted interactions between the immunoreagents and the endogenous tissue components. Avidin-biotin-based systems, for example, may cause unwanted staining by reacting with endogenous biotin. Another example is the presence of the endogenous enzyme, which tends to cause nonspecific chromogen precipitation. To avoid some of these problems, Mangham and Isaacson (1999) introduced a novel and sensitive peroxidasebased immunohistochemical detection method that employs mutually attractive, mirror image complementary antibodies (MICA). Such antibodies consist of two polyclonal antibodies raised in different species that are mutually attractive, i.e., they are raised against each other’s immunoglobulin species. Thus, each antibody is both an antigen and an antibody with respect to the other. Compared with the ABC technique, the MICA method allows up to 200-fold dilution of the primary antibodies with equivalent or superior immunostaining and shorter durations of incubation. Other advantages of the MICA method are that it is avidin-free and thus avoids nonspecific staining due to endogenous biotin, and yields ~64-fold increase in sensitivity (as judged by dot-blot) compared with that of the ABC technique. The improved sensitivity of the MICA protocol is thought to be due to increased stability of the complexes produced and possibly to antigen bridging. A minor limitation is the longer duration required to complete this method.

Procedure
Freshly paraffin-embedded or archival paraffin-embedded tissues can be used (Mangham and Isaacson, 1999). Sections ( thick) are deparaffininzed in xylene and rehydrated in descending concentrations of ethanol. Antigen retrieval is carried out by heating the sections in 1 mM EDTA (pH 8.0) in a pressure cooker at full pressure for 2 min. The sections are allowed to cool and are then transferred to TBS (pH 7.4). They are treated with 3% in methanol for 15 min to block endogenous peroxidase activity and washed in TBS/Tween ( Tween/1 ml TBS). The sections are incubated in the primary antibody (diluted in TBS) for 1 hr and then washed in TBS/Tween. They are incubated in 1:30 diluted link antibody (sheep antimouse Ig or sheep antirabbit Ig) (the MICA target antibody) for 20 min, and then washed in TBS/Tween. The sections are incubated in 1:30 diluted MICA antibody No. 1 (peroxidaseconjugated donkey antisheep Ig) for 20 min and washed with TBS/Tween. They are incubated in 1:30 diluted MICA antibody No. 2 (sheep antidonkey Ig) for 20 min and washed in TBS/Tween. This is followed by incubation with 1:30 diluted MICA antibody No. 1 (peroxidase-conjugated donkey antisheep Ig) for 20 min and washed in TBS/Tween. The sections are exposed to 1 mg/ml DAB-0.02% in TBS for 6 min and washed in TBS. They are dehydrated in ethanol, counterstained with hematoxylin, and cover-slipped.

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All the reagents are commercially available in kit form (polyMICA: Binding Site Ltd., Birmingham, UK).

FIXATION OF FROZEN TISSUES
Occasionally, tissues frozen for intraoperative consultation are processed for immunostaining. This situation arises, for example, when lesional tissue or atypical cells that are seen in a frozen section require confirmation. Some antigens show decreased or negative staining in tissues that are frozen before fixation with formalin, whereas the staining of some other antigens remains unchanged. For example, the staining of S-100, HMB45, synaptophysin, and neuron-specific enolase was negative in frozen tissues that were subsequently fixed with formalin, but the staining of these antigens was positive in freshformalin-fixed tissues (Edgerton et al., 2000). The staining of chromogranin was decreased in frozen-fixed tissues. In contrast, the staining of cytokeratins remained unchanged in frozen-fixed tissues. This and other evidence indicates that sections of frozen tissue that have been subsequently fixed with formalin may show false-negative staining. Although the exact mechanism responsible for the above-mentioned false-negative staining is not known, it is likely that cell membranes are disrupted when frozen tissue is thawed during fixation. Such a disruption does not occur when fresh tissue is fixed. Thus, damaged membranes would facilitate antigen diffusion out of the nucleus and/or the cell. This problem is especially serious for antigens in neural tissues (Edgerton et al., 2000); therefore, caution is warranted in interpreting immunohistochemical results of tissues that are fixed preceded by freezing. It is recommended that when surgeons freeze the tissue specimen, they should also fix freshly cut specimens of the same tissue for comparative study. Use of frozen sections without postfixation also has limitations in certain cases. The following example testifies to the drawback of using such sections for diagnostic purposes. Although intraoperative frozen-section evaluation for the surgical treatment of Hirschsprung’s disease is a common practice, a high rate of incorrect diagnosis of this disease has been reported using frozen sections (Maia, 2000). When surgical pathologists use primary resection without prior colostomy or frozen sections as the initial diagnostic test, the results of an incorrect frozen section could be disastrous. Because the concordance rate for frozen-section diagnosis on initial pathological specimens is low (67%), establishing an initial diagnosis of Hirschsprung’s disease on frozen sections is not recommended. Furthermore, the introduction of artifacts tends to make interpretation of already subtle histological findings untenable. It is therefore recommended that well-prepared permanent sections be used to establish the absence of ganglion cells in a rectal biopsy for the presence of this disease. It is known that pathological diagnosis of Hirschsprung’s disease is established by demonstrating the absence of ganglion cells in the colonic neural plexuses.

HOT SPOTS (AREAS) IN MICROWAVE OVEN
Microwaves consist of electric and magnetic fields, and they propagate in space. Electric fields are primarily responsible for physical effects on the tissue. The energy distribution and thus the speed of absorbing energy and warming up vary topographically. In

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other words, the spatial energy distribution in a microwave oven is unequal. A region in the oven with a high intensity of electromagnetic fields is known as a hot spot or, more correctly, a hot region (~1 cm). Hot areas are not located at fixed coordinates in the oven and are influenced by the load placed in the oven. Loads of various sizes and shapes lead to different heating patterns. By varying the position of the load within the oven during irradiation, reproducible results can be obtained. The problem of hot areas can be solved by providing a microwave transparent rotating platform for the load during irradiation and by placing an extra load to serve as a heat sink; a jar containing 100 ml to 1 liter tap water or antigen retrieval solution suffices. To obtain reproducible results the same type of jar should be placed in the same location in the oven. For detailed theoretical and practical considerations of hot areas, the reader is referred to Kok et al. (1993).

PROBLEM OF ANTIGEN RETRIEVAL STANDARDIZATION
Lack of reproducibility of results in epitope retrieval immunohistochemistry is a serious problem unacceptable in diagnostic immunopathology. Variable intra- and interlaboratory results are a common phenomenon. Lambkin et al. (1998) have assessed immunohistochemical results of estrogen receptor obtained from 16 Irish histopathology laboratories. They confirmed that although the majority of participants achieved acceptable immunopositive staining in the supplied sections, variations in the intensity of immunostaining, focal staining, and nuclear staining were observed. Battifora (1998) has discussed the necessity of minimizing at least intralaboratory variability of results, which is quite feasible. Both Lambkin et al. (1998) and Battifora (1998) have suggested means by which some degree of reproducibility of results can be obtained. A large number of factors influence the final results of immunostaining:
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
11. 12. 13.

Fresh or archival tissue specimens Type of fixative and its pH and concentration Duration of fixation, especially for formalin-sensitive epitopes Duration of tissue storage in formalin Embedding procedures Type of retrieval fluid and its pH Type of microwave oven and heating parameters, other heating sources, or other epitope retrieval treatments such as enzyme digestion and ultrasound Monoclonal or polyclonal antibodies Tested or new antibodies obtained from the same clone or not Source of antibodies; even similar monoclonal antibodies obtained from different sources may show differential affinity for the epitope Incubation times and temperatures Variable staining methods; automated immunostainers or manual staining Differences in the interpretation of results

Considering the processing variables enumerated above and below, achieving complete standardization of immunoreactions is extremely difficult. Standardization of fixation is difficult due to the large number of variables, including the volume and concentration of

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the fixative, temperature and duration of fixation, size and composition of the tissue, and the amount of free blood. Also, irrespective of the size of the tissue block, the fixation is variable within the block. The same duration of fixation with formaldehyde may or may not result in identical fixation, for each tissue block responds uniquely to the fixative. Moreover, uniform sections are difficult to obtain, and their thickness is difficult to determine. Many sections are wedge-shaped rather than planoparallel (Rittman, 1998). In addition, the arc of vibration caused by the knife edge as it cleaves the section is sufficient, for example, to glide over or under the surface of some nuclei or cut through the remainder. Consequently, an accurate measurement of the concentration of nuclear antigens is difficult (Allison, 1999). The tendency is to cut the thinnest possible sections to obtain superior resolution that provides distinct images. However, thin sections show excessive compression as well as variation in thickness because compression is usually inversely proportional to section thickness. Another obstacle is the small number of sections that are usually examined in a diagnostic laboratory, limiting the production of reliable average or quantitative data.

TEST BATTERY
A standardized method for retrieving a given epitope in a particular tissue can be developed by using the test battery approach (Shi et al., 1996a). This is a convenient and rapid means to optimize three important factors (pH, temperature, and duration of heating) responsible for the immunostaining of a given epitope-antibody combination. The optimal protocol lessens false-negative immunostaining. The need for this approach arises when optimal conditions for retrieving an epitope are not known. It is known that the retrieval of different epitopes requires specific retrieval conditions. These conditions primarily consist of the pH and the temperature of the epitope retrieval fluid in the microwave oven and the duration of heating. Other factors influencing the final results of immunostaining are not included in this test. The following three levels of heating durations and three pH levels of the epitope retrieval fluid (sodium citrate buffer and Tris-HCl buffer) have been recommended to determine the optimal protocol for the retrieval of an epitope (Shi et al., 1996a).
Temperature and duration Buffer pH

120°C for 10 min 100°C for 10 min 90°C for 10 min

pH1–2 Slide # 1 Slide # 2 Slide # 3

pH 6–8 Slide # 4 Slide # 5 Slide # 6

pH 9–11 Slide # 7 Slide # 8 Slide # 9

One reason for interlaboratory differences in the reproducibility of immunostaining results is the use of microwave ovens with significant differences in age, power, construction, and design. Individual laboratories should optimize wattages and duration of heating as well as durations of each of the steps mentioned above; some of them will need to be determined by trial and error. In the absence of standardization, since either false-positive or false-negative immunostaining can occur with any antigen retrieval protocol, the effect of the chosen antigen unmasking method on every individual antigen must be determined using careful

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controls. Appropriate positive and negative controls, as well as the study of fresh-frozen tissue sections, are required to rule out any false-negative or false-positive staining. Duplicate immunostaining will assess reproducibility.

INTRAOBSERVER AND INTEROBSERVER VARIATION IN DIAGNOSIS
Pathologists play a key role in the diagnosis of cancer, and their histopathological assessments are accepted as the gold standard. Although not admitted, the process by which a pathologist makes a diagnosis is inherently subjective. A number of factors, including clinical features of the lesion, the clinical impression offered by the surgeon, and the training and experience of the pathologist, play a part in determining the final “signout” diagnosis on which the final treatment decisions depend. These decisions have farreaching consequences in the quality of health care. It is obvious that no other type of error in the medical profession is more important, less understood, and less frequently admitted than fallibility in histopathological diagnosis. Kaugars (1995) has aptly pointed out that the sign-out is written on paper, not on stone tablets. Can the intraobserver and interobserver variations in diagnosis be eliminated? Unfortunately, the answer is no. However, avoidable fallibilities must be avoided. The interobserver variations can be significantly reduced by a joint session behind a microscope in a process of “practical agreement” (Vet et al., 1995). Prior to such sessions, participating pathologists reach consensus on the relevant pathological grading characteristics (theoretical agreement). A theoretical agreement increases the practical agreement between pathologists. Interobserver agreement is definitely improved when pathologists confront each other’s observations and arguments. Even experienced pathologists will benefit by a joint session behind the microscope. Both low-power and high-power microscope observations are useful in markedly minimizing interobserver variation. The characteristics observed at low magnification include atypia, location of immature cells, and stratification/polarization. At high magnification, detailed morphological characteristics, such as location of immature cells and stratification/ polarization (differentiation), nucleus/cytoplasm ratio, hyperchromasia, polymorphous nuclei (cell characteristics), and the location and appearance of mitotic activity, are scored (Vet et al., 1995). Another approach to avoiding interobserver and intraobserver variations and standardizing the diagnosis is the use of computer-assisted analyses. This technology is beginning to be employed in some laboratories (see below).

QUANTITATION OF IMMUNOSTAINING
Accurate quantitation of antigens using immunohistochemistry depends upon a linear relationship between the amount of antigen and the intensity of immunoperoxidase-DAB reaction product as well as the percentage of stained cells. Variations in staining intensity will reflect the amount of antigen only if optimal preparatory procedures are used; for example, oversaturation of the chromogen reaction may result in invalid quantitation. Therefore, optimal concentration of DAB should be determined by trials with DAB

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concentrations ranging from 0.1–1.5 mg/ml in Tris buffer containing 0.003% (pH 7.6). Optimal duration of DAB incubation should also be determined by trying incubations for 3–15 min at 37°C. Currently, interpretation of immunohistochemistiy staining in most studies is subjective, qualitative, and nonreproducible. The achievement of quantitative, reproducible results requires standardized immunohistochemical procedures. The development of such a universal procedure is difficult because antigens are not equally affected by a specific processing protocol, including fixation. However, instruments to computerize image analysis have the potential to quantitate immunohistochemically localized antigens. One such instrument is Cell Image Analysis System SAMBA 4000 (Imaging Products International, Chantilly, VA) used in combination with the automatic stainer (OptiMax Staining System, BioGenex Lab., San Ramon, CA) or Tech Mate (Biotek, Santa Barbara, CA). The image analyzer contains software for the densitomeric and RGB (red, green, blue) to HSI (hue, saturation, intensity) colorimetric analysis of cells and tissues (Esteban et al., 1994a). It is based on a light microscope attached to an interactive microcomputer that is capable of high-speed digital image processing for cell measurements. The system includes various software packages for different applications (De Cresce, 1986). It allows immunostained histological sections to be represented as digitized images from which the optical densities of the DAB reaction product over a specific cell part or component (e.g., nucleus) can be quantitated. Bacus et al. (1988) have successfully quantified the estrogen receptor content in human breast tumors. The data showed excellent sensitivity and specificity. Quantitative immunostaining analysis has been performed with a computerized microscopic image processor, SAMBA 200 (SAMBA TITN, Grenoble, France) (Seigneurin et al., 1987). Integrated optical density histograms are provided by the image analysis processor. Mean values and percentage of immunostained cellular surfaces are computerized by the application processor. These computerized systems facilitate multiparametric, accurate, reliable, reproducible, and automatized evaluation of the heterogeneity of the antigenic sites in tumors (Charpin et al., 1989). The advantage of this capability becomes apparent when one considers that tumors showing positive immunostaining are pools of positive and negative cells. To rectify the lack of intra- and interlaboratory reproducibility of immunostaining, Riera et al. (1999) have described the Quicgel method used in conjunction with computerassisted image analysis for quantitation of immunohistochemical data. The Quicgel consists of a cultured cell plate containing a known amount of the antigen, which yields consistent positive staining detected by image analysis. The Quicgel is processed simultaneously with the specimen. This method is based on the assumption that changes in the antigen content of the Quicgel and the specimen vary in parallel during specimen processing. Thus, a decrease in the immunostaining of the specimen during processing is equally demonstrated in the Quicgel. Even without image analysis, Quicgel can serve as a control in immunohistochemical staining. Further application of this protocol for quantitation is awaited. A related approach was used by Ranefall et al. (1998) to quantify images of immunohistochemically stained cell nuclear Ki-67 antigen and cyclin A protein in bladder carcinoma tissue. They combined automatic, computerized image analysis with appropriate controls and reference material. This approach is superior to the automatic method without

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controls. The former method consists of simultaneous processing of embedded cultured controlled cells and tissue sections. Agarose-embedded cultured fibroblasts are fixed, embedded in paraffin, and sectioned at They are immunostained together with paraffin-embedded tissue sections. The image of the control cells serves as a standard control regarding image qualities such as illumination and color properties. Since control cells possess known characteristics regarding antigen expression to be examined in tissue sections, they serve as a means to control and standardize immunohistochemical data (Ranefall et al., 1998). Recently, an automatic color video image analysis system was developed to quantify antigen expression (androgen receptor) (Kim et al.,T999a). This system provides a linear relationship between the antigen content and mean optical density of the immunoperoxidasesubstrate reaction product. Titration of antibody, concentration, and reaction duration of the substrate can be optimized with this system. The imaging hardware consists of a Zeiss microscope, a three-chip charge-coupled-device camera, a camera control board, and a Pentium-based personal computer. It is necessary to properly maintain and calibrate the computerized image analysis system. This is the only way to ensure that the scale of the reported histogram will cover the range of staining intensities obtained in practice (Bacus et al., 1988). In addition, the technician should be knowledgeable about the normal cellular morphology and pathology of the tissue. Counterstaining tissue sections with methyl green in conjunction with the chromogen is also necessary. A relevant question is whether the quantitative measurements obtained with currently available computerized image analysis systems are reliable, accurate, and reproducible and if quantitation of immunostaining reactions offers any real advantage over qualitative evaluations by an experienced pathologist (Rittman, 1998). Many of the image analysis systems are still somewhat rudimentary. Results obtained with various image analyzers are difficult to compare because different hardware and software are used. Unfortunately, in some laboratories quantitation of immunohistochemistry may be used simply to justify the pathologist’s decision in difficult (borderline) cases. The controversy over whether borderline tumors should be classified as benign or malignant and whether they represent a precursor of malignancy remains unresolved. A semiquantitative approach has also been applied for evaluating the concordance between the presence of p53 mutations and immunohistochemical overexpression of this protein in breast carcinomas (Schmitt et al., 1998). The advantage of this approach is that it uses a scoring system based on both the intensity and percentage of stained cells. A limitation of this method is that scoring is performed by examining all low-power optical fields containing tumor, which is time consuming, lacks automation, and is thus subjective, even when scored by more than one observor.

AUTOSTAINERS
Presently, immunohistochemistry requires improvements in quality, reproducibility, speed, quantitation, and standardization. Some of these goals can be achieved by using computerized bar code–driven automatic immunostainers that automatically dispense reagents, control washing, mixing, and heating to optimize immunohistochemical reaction

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kinetics and that produce results within 1 hr. Furthermore, the practice of manually staining a large number of slides is tedious and time consuming. Typically, manual hematoxylin-eosin staining (hematoxylin stains nuclei blue and eosin stains cytoplasm pink) is completed in approximately 25 steps. Autostainers save a technologist’s time, which permits him or her to carry out more technically demanding tasks. After the staining protocol has been standardized, the stainer does not require the technologist’s attention during its operation. There are many other advantages to using autostainers. They prevent risk of exposure to certain hazardous reagents (xylene). Some stainers have built-in fume hoods or can be used in a fume hood; in the former case, the need for a large overhead fume hood is eliminated. Another advantage is consistency of the technique that eliminates intra- or interpersonal variations in results. Also, a consistent temperature can be maintained for temperature-sensitive procedures. The space requirement for some stainers (e.g., centrifugal stainer) is smaller than that required for manual staining for some procedures. Efficient stainers use reagents conservatively, thus reducing the amounts needed and the risk of contamination. There are a few limitations to equipping a laboratory with an autostainer. The high cost of most stainers may be prohibitive for a small laboratory. Unavailability of space in such a laboratory is also a possibility. Repairs of this machine are expensive and may take a long time if the service technician lives in another state. Some stainers require the purchase of prepackaged reagents that are also relatively expensive. Moreover, some technologists may object to being restricted to using these reagents. In addition, a staining defect occurring during operation of the stainer is revealed only after staining is complete. On the other hand, if a problem arises during manual staining, the process can be stopped and the problem corrected. But above all, the use of a stainer limits the technologist’s understanding of the actual staining process. There are two types of autostainers: in the first type, the slides are immersed into the reagent; in the second type, the reagent is applied to the slides. Stainers that immerse the slide into the stain (bath stainers) can be either linear or batch design (Earle, 2000). The linear type is based on a carrier mechanism that allows loading of the slides into the slide holders (racks), one at a time, and their sequential immersion into the staining solution. The slide holders are attached to the carrier, which moves at a uniform speed, and the slides exit the stainer one at a time. This type of machine is long, processes ~360–720 slides per hour (12–14 min per slide), and requires water and a drain. It may have a built-in fume hood and slide dryer. Batch stainers move slide holders, each containing several slides, through baths of the staining solution. Programmable batch stainers are now commercially available which use robotic arms to move the slide racks from one position to the next. These stainers can be programmed to agitate the slides in the staining bath. Simultaneous multiple staining can be accomplished in some machines based on this principle. A combined linear-batch stainer is also available, which moves slide containers through a series of stain containers. Each rack may hold a small or large number of slides, and continuous staining is possible. The machine is long, processes 24–66 slides per rack, and the time taken depends on the program. It may be compatible with the coverslipper, and some have built-in fume hoods. The machine may require running water and a drain, and it may have waste collection. There are three types of stainers that apply the reagents to the slides: capillary gap stainers, centrifugal stainers, and flat-method stainers (Earle, 2000).

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Capillary Gap Stainers
Capillary gap stainers are based on the principle that the staining solution is forced between the slide and the area around it. Essentially, rotating gears move slides (face downward) along a plane surface that has holes through which the stain is pumped at appropriate intervals. The advancing slides press a switch as they pass each staining station, thus activating a pump. The stain is discarded after the slide moves to the next station, avoiding the contamination of the bulk containers. The machine pumps the stain from the closed bulk containers to the plane surface via small tubing, minimizing reagent evaporation. The capillary gap system can also be used in stainers that use two slides face-to-face to provide the capillary gap. Robotic arms move holders of paired slides to staining, draining, and rinsing stations. Because this system uses very little staining solution, it is recommended for immunostaining large numbers of slides. The machine is ~3 ft long, may be compatible with a cover-slipper, and some may have a built-in fume hood. This system requires prepackaged reagents and may require a drain.

Centrifugal Stainers
Centrifugal stainers spray the staining solution onto the slides as they rotate past the spray nozzles in a spinning chamber. The prepackaged reagents are in closed containers with pump tubing, which prevents evaporation and contamination of the chemicals. The machine is smaller than 2×2 feet, stains 12 slides in 6–8 min, requires prepackaged reagents, and may require a drain. It usually does not require a fume hood.

Flat-Method Stainers
Flat-method stainers drop staining solutions onto the slide as it lies flat within the stainer. Some stainers employ robotic arms to apply solutions to the slides. This system is in common use for immunohistochemical staining. The machine is long, stains 20–40 slides, depending on the system, in and slides require predeparaffinization. The protocol may require prepackaged or manufacturer’s reagents and a waste container. It is recommended for immunohistochemistry. For additional details about autostainers, see Earle (2000). The following automatic tissue processors and stainers are commercially available.
1. AP 280 Embedding Station, Carl Zeiss, Inc. One Zeiss Drive, Thornwood, NY 10594 2. ATP1 Tissue Processor, Triangle Biomedical Sciences, Inc. 3014 Croasdaile, Durham, NC 27705–47770 3. Cytologix Stainer, Cytalogix Staining System 99 Erie Street, Cambridge, MA 02139 4. Lab Vision Auto Stainer, DAKO Corporation, 6392 Via Real, Carpinteria, CA 93013

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5. Mark 5 HSS Stainer, Diagnostic Products Corporation 5700 West 96 Street, Los Angeles, CA 90045 6. Medite TST40 Slide Stainer, Mopec 13640 Elmira Street, Detroit, MI 48227 7. Medite TPC15 Tissue Processor, Mopec 13640 Elmira Street, Detroit, MI 48227 8. Optimax Consolidated Staining System, Biogenex Laboratories 4600 Norris Canyon Road, San Ramon, CA 94583 9. Protocal Capillary Action Stainer, Biochemical Sciences, Inc. 200 Commodore Drive, Swedesboro, NJ 08085 10. Tissue-Tek. DRS 2000, Slide Stainer, Sakura Finetek USA, Inc. 1750 West 214 Street, Torrance, CA 90501 11. Ventana ES Automated Immunostainer, Ventana Medical Systems, Inc. 1910 Innovation Park Drive, Tucson, AZ 85737

VOLUME-CORRECTED MITOTIC INDEX
The volume-corrected mitotic (M/V) index can be used to test for differences between borderline and malignant tumors. This index expresses mitotic activity as the number of mitotic figures per square millimeter of neoplastic tissue in the microscope field. Usually 10 fields are counted at a magnification of 40, which corresponds to of neoplastic tissue in the section. The M/V index has the advantage of not being influenced by the size variation of the microscope field or cellularity of the neoplasm (Haapasalo et al., 1989). Also, this method is easy, relatively rapid, reproducible, inexpensive, and available to all pathologists (Miliaras, 1999). The morphometric formula of the M/V index renders mitotic counts a more reproducible criterion because it avoids some of the limitations, such as differences in microscope field size, of the conventional mitotic index. The M/V index has been used for mitotic counts in many human neoplasms for both diagnostic and prognostic purposes (Lipponen et al., 1990). Recently, Miliaras (1999) has used this index for determining differences in p53 immunoreactivity and the proliferation rate between borderline and malignant ovarian tumors. The M/V index is evaluated as the number of mitoses per 10 hpf (high power field) and is calculated according to the following formula proposed by Haapasalo et al. (1989).

Where n=number of microscopic fields studied (usually 10) Vv=volume fraction of neoplastic tissue (%) as expressed in the area fraction of neoplastic tissue in the microscope field; this is estimated subjectively in the same field in which the mitotic count is made. MI=number of mitotic figures in a microscopic field from the area of highest neoplastic cellularity (during the measurement, the microscope is focused only once). k=coefficient characterizing the microscope: where r is the radius of the microscope field in millimeters (0.255 mm).

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THE GLEASON GRADING SYSTEM
Gleason grading is now the most widely used system for grading prostatic carcinoma. This system is an effective tool for prognostication and as an aid in therapeutic decisions for men with prostate cancer. The system is characterized by two major features: (1) it is based solely on architectural pattern but cytological features are not evaluated (Gleason and Mellinger, 1974) and (2) the overall grade is not based on the highest grade within the tumor. The prognosis of prostate cancer is intermediate between the most predominant and the second most predominant pattern of cancer (Fig. 5.3). Consequently, the grades of the most prevalent and the second most prevalent pattern (~5% of the tumor) are added together to obtain a Gleason score (Allsbrook et al., 1999). If the tumor shows only one pattern, the pattern grade is doubled to obtain the Gleason score; for example, for all pattern 3, the Gleason score is 6 (Fig. 5.4). The Gleason score is directly correlated with mortality rates, is a predictor of time to recurrence after surgery, and of response to therapy. Presently, the Gleason score, along with PSA and tumor stage, forms the database upon which radical therapies are recommended (King, 2000). The Gleason score alone suffers from interpretation bias and its accompanying grade errors. Evidence is available indicating a lack of interobserver reproducibility of this score. As expected, interobserver agreement is significantly better among pathologists who learned Gleason grading at a professional meeting or course than among those who had

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not (Allsbrook et al., 2001a, b). The interpretation bias is significantly minimized through a consensus pathological evaluation, while sampling effects are maximally reduced by using an optimal number of biopsy cores. These two remedies, when applied in combination with the Gleason score, result in maximal grading accuracy. Another approach to

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achieve grading accuracy is through using cytokeratin staining in combination with a consensus of three pathologists (Carlson et al., 1998). Although the use of sextant biopsy is an effective technique for prostate cancer diagnosis (Stamey, 1995), and has now become the most common method, it has limitations. Studies have shown that grading based on sextant biopsies, when compared with matched surgical grades, suffers from a significant rate of undergrading. Therefore, biopsy cores should be outside of the anatomical domain of extant biopsies to reduce missed or delayed diagnosis (King and Long, 2000). Because prostate cancer is often multifocal (heterogeneous population of tumor cells), a certain degree of sampling error is expected. The error may result from sampling an area which is either overrepresented with high-grade tumor or, conversely, overrepresented with low-grade tumor as compared to the actual histological grade of the resected prostate (King and Long, 2000). It is not uncommon for well-differentiated cancers to be undergraded and poorly differentiated cancers to be overgraded. To overcome such sampling errors, a directed biopsy must be performed (assuming that an ultrasound-visible lesion is present) or the number and location of biopsies must be increased. In conclusion, the grading error can be significantly reduced by minimizing sampling effects through increasing the number and location of biopsies. The positive role of consensus pathological evaluation in lessening grading errors is equally important. These two remedies will improve the accuracy of Gleason grading of prostate biopsies. As stated above, most patients with prostate carcinoma are diagnosed by core needle biopsy, and tumors are most commonly graded using the Gleason grading system. The Gleason score assigned by the pathologist to the tumor obtained by needle biopsy can profoundly affect the treatment decisions made by urologists, radiation oncologists, and medical oncologists. Although presently the Gleason grading is in common use, many studies indicate inaccuracies in this grading system, with a strong tendency toward undergrading (Carlson et al., 1998). To improve the accuracy of the Gleason grading system, Kronz et al. (2000) developed a free, web-based tutorial program (www.pathology.jhu.edu/prostate). It consists of 20 pretutorial quiz images of prostate carcinoma specimens that were obtained by needle biopsy for grading, followed by 20 tutorial images with text describing the Gleason grading system. Subsequently, pathologists take a posttutorial quiz, consisting of the same 20 images that were used in the pretutorial quiz. This web site tutorial leads to an improvement, especially in the grading of high-grade tumors (Gleason score, 8-10). The web site also improves the grading of tumors with Gleason scores of 5–6 on needle biopsy. An advantage of the web-based media is that it is permanently available for repeated review as opposed to other learning experiences such as lectures.

UNIVERSAL ANTIGEN RETRIEVAL METHOD?
Is it practical to develop a universal epitope retrieval method? The answer is no, as the optimal retrieval of each type of epitope requires very specific processing conditions such as fixation, retrieval fluid, and unmasking treatments, including heating, enzymatic digestion, and ultrasound. In terms of preservation and masking, each type of epitope is affected differently by the fixative used, and by its concentration, pH, and the temperature and duration of fixation. As long as different fixatives are used in different laboratories,

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interlaboratory standardization of immunohistochemistry will remain elusive. Storage of tissue in the fixative, as well as sections of the fixed tissue mounted on glass slides, influences epitope retrieval. Some evidence indicates the potential loss of immunostaining in stored paraffin sections, especially with prolonged durations at room temperature (see page 84). It is not uncommon for pathology laboratories of small hospitals to mail unstained slides to other laboratories for immunostaining and second opinions. Epitope retrieval depends upon epitope preservation and hence on the effects of fixation or chemical fixation. In some cases, the fixation history of archival tissues is not known, making the rational choice of epitope retrieval methods problematic. Another problem is that similar antibodies obtained from different sources may vary in their sensitivity and specificity. The procedure used to dry sections on a glass slide may also affect the degree of immunoreactivity. This is exemplified by nuclear antigens (e.g., proliferating cell nuclear antigen) that show decreased immunoreactivity when sections are hot-plated onto glass slides (Hall et al., 1990). The composition, pH, and amount of retrieval solution influence the degree of epitope retrieval. In addition, the type of heating used (microwave, autoclave, steamer, hotplate, and conventional oven) affects the degree of epitope retrieval. Overwhelming evidence indicates that all types of epitopes are not equally unmasked with any one source of heating. In other words, certain types of epitopes are maximally retrieved with microwave heating, while some other types are best retrieved with an autoclave or a steamer. In addition, ultrasound treatment may be efficient for unmasking certain epitopes. Furthermore, optimal retrieval of certain epitopes is obtained with combined treatments such as microwave heating-ultrasound or enzymatic digestionmicrowave heating. Caution is warranted in the use of enzymatic digestions, as this treatment can adversely affect cell morphology and antigenicity. The temperature used during epitope retrieval is important, and if microwave heating is used, so is the amount of water load and its exact location in the oven. The extent of epitope retrieval is also section-thickness dependent. It should be noted that the same processing conditions show different retrieval efficiencies of similar epitopes in animals of different species and ages. Whether a protein molecule is glycosylated or not affects its unmasking with an epitope retrieval protocol. For example, it has been demonstrated that the more glycosylated human placenta fibronectin has a higher resistance to protease treatment, and thus a reduced epitope retrieval, than the less glycosylated plasma fibronectin (Zhu et al., 1984). Even different isomers of an antigen may require different epitope retrieval methods. In addition, the degree of epitope retrieval is ethnicity-dependent. Also, optical microscopy, fluorescence microscopy, and electron microscopy require different methods of fixation and epitope retrieval. A pretreatment that facilitates the recognition of a given epitope may destroy other epitopes in the same antigen or in other antigens. In conclusion, although a universal epitope retrieval method is almost impossible to formulate, the development of a general strategy for adequate retrieval of epitopes is feasible. Such an approach is presented in Chapter 8.

CALIBRATION OF MICROWAVE OVEN
As stated earlier, lack of a standardized antigen retrieval method results in intra- and interlaboratory variability in immunostaining results. The following method of microwave

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oven calibration is a step toward obtaining reliable immunostaining (Tacha and Chen, 1994). This approach avoids repeated interruption of oven heating to replenish the antigen retrieval fluid. The method essentially establishes the time to boiling point, after which the setting is adjusted to maintain a simmering temperature. At such mild temperatures, the separation of sections from the slide is less likely. Place the jar containing 250 ml of antigen retrieval fluid and slides in the center of the microwave oven, and set the oven on high power (800 W) for 2–3 min, until the fluid begins to boil. Turn off the oven and record the exact time it took to achieve boil. Set the oven on low power (~300 W) for 7–10 min, and adjust the setting so that the oven cycles on and off every 20–30 sec and the fluid boils for ~5–10 sec/cycle. Also, note this setting. The following formula can be used to determine the power setting: S = 250/P × 10, where S is the oven power setting and P is the output power of the oven. For example, if the oven output power is 800 W, the power setting for antigen retrieval (S) will be S= 250/800 × 10= 3.1. Therefore, set the oven on 3 and heat at 100°C for 7–10 min to achieve antigen retrieval, depending on the antibody used. Microwave ovens with temperature readouts are commercially available (Energy Beam Science, Agawam, MA). Their power output is regulated by a temperature feedback mechanism and timer, so that both temperature and time can be monitored. They can also be used for fixation and accelerated immunostaining.

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Chapter 6

Antigen Retrieval

POSSIBLE MECHANISMS OF ANTIGEN RETRIEVAL
Heating, especially microwave heating, effectively unmasks a wide variety of epitopes (e.g., Fig. 6.1). However, in some cases epitopes are best unmasked by methods other than heating. Such alternate procedures include enzyme digestion and treatment with detergents. Furthermore, the retrieval of some epitopes is accomplished with a combination of methods such as microwave heating–enzyme digestion, microwave heating–sonication, or microwave heating–EDTA. In fact, variations of the heating method may affect epitope retrieval differentially. These variations include microwave heating, autoclaving, pressure cooker, water bath, and even a hot plate. Comparative studies using different heating methods demonstrate that an antigen under study is retrieved optimally by only one of the heating variations, and that heating method may be other than microwave heating (see pages 153–154). However, it is most likely that each heating method is equally effective in antigen retrieval provided it is used under its optimal conditions. Nevertheless, microwave heating is presently the most commonly used procedure to retrieve antigens in formalin-fixed tissues. These observations suggest that multiple mechanisms are responsible for epitope retrieval. A number of possible mechanisms responsible for epitope unmasking have been advocated, including breakage of protein crosslinks introduced by formaldehyde, denaturation of proteins to reveal previously masked epitopes, and unmasking of epitopes by removing calcium ions. Major mechanisms are discussed below. Fixation with formaldehyde tends to alter the conformation of the protein molecule, making it unrecognizable by the antibody. Antigen retrieval treatments may restore the original, native protein structure, reestablishing the three-dimensional structure of the protein, or coming very close to that state (Shi et al., 2001). In other words, antigen retrieval treatments may renature the protein structure that was altered during fixation. However, direct evidence supporting the revival of the native conformation of the epitope with antigen retrieval pretreatment is lacking. On the other hand, available evidence supports the occurrence of the breakage of protein crosslinks, which allows the antibody access to the antigen. Conventional heat, dry or steam, breaks down reversible protein-protein, protein–nucleic acid, and protein-carbohydrate crosslinks introduced by formaldehyde and thereby unmasks the epitopes, as well as allowing the antibodies access to the epitopes. It is well known that most, if not all, crosslinks formed during formaldehyde fixation are destroyed upon heating even at 37°C
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for a prolonged time (e.g., 48 hr) or at higher temperatures (e.g., 80–100°C) for very short durations, that is, in minutes. Specific peptide-bond cleavage by microwave heating in weak acid solutions is a well-established method in protein chemistry (Wu et al., 1992). The specific cleavage sites of peptide bonds are located at the carboxyl- and amino-terminal ends of aspartyl residues along the peptide chain. Thus, heat can free epitopes from other proteins and attached molecules. That heat is not the only method to unmask epitopes is exemplified by enzyme digestion or detergent treatment. The exact mechanism responsible for epitope retrieval with ultrasound is not clear, although intense heat is produced for an exceedingly short duration. It is known, however, that ultrasound and/or heat decreases the amount of negative charges on the cell surface (Joshi et al., 1983; Adler et al., 1988). Mechanical vibrations of molecules caused by ultrasound and heat are thought to unfold the protein molecule and to expose the epitopes. The mechanism underlying unmasking of epitopes with digestive enzymes is better understood. Enzymes such as trypsin II, used in epitope retrieval, are powerful, tested protein-digestive molecules. They are known to digest proteins and break down protein crosslinkages introduced during formaldehyde fixation. As a result, the tight network surrounding the epitopes is dismantled, allowing access of antibodies to the epitopes. If antigen retrieval with protease digestion must be carried out, 100 mg of trypsin in 100 ml of Tris-buffered saline (pH 7.8) can be used for 15–20 min at 37°C.

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Some information is available on the possible mechanism underlying masking of epitopes in the presence of calcium ions. Such a masking of epitopes and their unmasking are discussed in detail on page 120.

Nonthermal Effects of Microwave Heating
Like conventional heat, microwave heat breaks down protein crosslinks. The most common explanation presented in the literature for unmasking epitopes with heat in the microwave oven is hydrolysis of protein crosslinkages. However, the effects of microwave heating on the tissue sections resulting in epitope retrievals are exceedingly complex. Heat alone may not be enough to explain the effects of microwaves on epitope retrieval. This view is supported by the observation that microorganisms are killed at lower microwave temperatures or with shorter exposures than those required when conventional heat is used (Chipley, 1980). Furthermore, because microwave heating also enhances immunostaining of ethanol-fixed tissues, it is apparent that such heating unmasks epitopes by a mechanism other than or in addition to breakage of protein crosslinks because this coagulative fixative does not introduce crosslinks. The above-mentioned evidence indicates that in addition to the direct thermal effect of microwaving, microwave heating facilitates epitope retrieval by another simultaneous mechanism. The microwave energy irradiated on the tissue sections in various liquid media is lost or absorbed by the samples by two mechanisms: ionic conduction and dipole rotation. Both effects occur simultaneously to account for the phenomenon of rapid heating (Kingston and Jessie, 1988). It is thought that microwaves unfold protein molecules, exposing the epitopes by subjecting the molecules, at least polar molecules such as water and polar side chains of proteins, to rotational movement. As a result, these molecules reach to a high energy level, unmasking the epitopes. It is known that microwaves interact with dipolar molecules by (1) imparting kinetic energy and raising temperature and (2) altering electric fields. Microwaves induce dielectric fields, causing dipolar molecules to rapidly oscillate 180 degrees. In other words, these molecules oscillate at the frequency of 2,450 MHz or at about 2.5 billion cycles per second. Thus, microwave action is also due to rapid oscillation along the axes of asymmetrical molecules such as water, proteins, and fatty acids, which behave as dipoles in an attempt to reorient their positive and negative poles to keep up with the rapidly changing electrical fields generated by microwaves (Salvatorelli et al., 1996). It has been shown that the oscillating electric field causes cell poration (Chang, 1989). It is also known that microwaves irreversibly alter the plasma membrane, with subsequent changes in ion transport, breakdown of hydrogen bridges and secondary bridges, alterations in protein hydration, and release of bound water. All of these phenomena explain why microwaves exert a different effect than that of conventional heat. It is apparent that not only the thermal but also the nonthermal component of microwaves deserve consideration as effective epitope retrieval factors. Evidence indicates that microwaves affect the kinetics of conformation changes of proteins such as (Bohr and Bohr, 2000). It is thought that even approximately a few GHz can excite protein molecules. Consequently, the kinetics of conformational changes of the protein molecule are enhanced, and this denaturing effect is

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nonthermal. In fact, microwave irradiation can cause folding or unfolding of protein molecules. However, additional evidence is needed to substantiate the role of the nonthermal effect of microwave irradiation in antigen retrieval.

EFFECT OF ENDOGENOUS CALCIUM ON ANTIGEN MASKING
An understanding of the interaction between the antigen (epitope) and the antibody visualized immunohistochemically can be attempted by considering at least the role of formalin fixation, antigen retrieval fluid, and heat treatment. How each of these three factors affects the molecular structure of the antigen or the epitope is the fundamental question. The significance and relevancy of this question are apparent because an antibody recognizes the corresponding epitope based on the molecular structure of the latter. The following discussion considers the possible role of calcium in masking antigens during fixation with formaldehyde; it is based primarily on studies carried out by Morgan et al. (1994, 1997a, b), Shi et al. (1997, 1999a), and Taylor et al. (1996a, b). The role of other factors in antigen masking and retrieval is discussed elsewhere in this volume. Endogenous calcium is an important factor in epitope masking. Biochemical studies indicate that calcium binding induces a conformational modification of the protein molecule, resulting in either a reduced antigen-antibody recognition effect (e.g., for thrombospondin) (Wilson, 1991) or the reverse effect (for protein C, a vitamin K–dependent enzyme involved in blood coagulation) (Wakabayashi et al., 1986). It has been proposed that removal of calcium by chelation significantly modifies the thrombospondin conformation (Dixit et al., 1986). These changes may expose epitopes necessary for the binding of certain monoclonal antibodies. This and other evidence indicates that calcium-induced changes in the conformation of different proteins may result in negative or positive detection of immunogenicity. The ability of some monoclonal antibodies, but not all, to recognize their corresponding epitopes is calcium-dependent under certain conditions. Different antibodies respond differently to the calcium-induced modification of the same protein. In relation to fixation with an aldehyde, possible mechanisms responsible for masking or unmasking epitopes as a result of tissue-bound calcium and calcium chelation, respectively, are detailed below. One of the major effects of formaldehyde fixation is the generation of a large number of hydroxymethyl groups through selective interactions with various functional groups (e.g., active hydrogens on aromatic rings, primary and secondary amines, and hydroxyl and sulfhydryl groups) in proteins. The hydroxyl component of the hydroxymethyl groups is thought to be reactive, depending on which of these functional groups the formaldehyde is bound to. Such an active hydroxyl component could form a coordinated bond with calcium ions (Fig. 6.2). Thus, proteins fixed with the aldehyde may become complexed with calcium ions that are abundant in animal tissues, reaching levels on the order of 2 mM in the cytoplasm of eukaryotic cells. These complexes mask epitopes to a variable degree. Calcium complex formation with proteins in this state is likely to be quite strong, involving four to eight coordinate bonds. Therefore, a considerable amount of energy (heat) is required to release the calcium ions from this cagelike complex. Based on this observation, calcium released from this complex requires hightemperature heating in combination with a calcium chelating and/or precipitating agent such as EDTA, EGTA, citrate buffer, or urea. Because these reagents are chelators of divalent

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metal ions, epitope unmasking can be achieved by exposing the sections to such treatments. Figure 6.3 shows the disruption of some coordinate bonds at a high temperature in the presence of EDTA, which results in antigen retrieval. Also, an inorganic salt such as sodium carbonate is expected to remove calcium by preferential precipitation. It is interesting to note that microwave heating also causes changes in metal ion transport through the plasma membrane.

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Further evidence supporting the role of calcium in antigen masking was provided by Shi et al. (1999a). They exposed frozen tissue sections to 50 mM (pH 7.1) overnight at 4°C and demonstrated a significant loss of immunostaining or altered staining pattern of antigens such as thrombospondin and Ki-67 compared to controls not exposed to Such a loss of staining can be partially recovered by incubating the sections in EDTA, substantiating the role of calcium in antigen masking. The antigen masking effect of calcium has also been demonstrated by adding to antigen retrieval fluid such as EDTA (Kim et al., 1999b). The disodium salt of EDTA (in common use) binds one divalent metal ion only, and the addition of molar excess of calcium ions would eliminate the antigen retrieval effect of EDTA. The role of pH in calcium-related effects on antigen unmasking is controversial. According to Morgan et al. (1997b), two different mechanisms are involved in antigen retrieval at acidic and alkaline pH levels. Under acidic conditions (pH 1–3), instead of chelation, high concentrations of hydrogen ions dissociate calcium complexes and/or breakdown protein crosslinkages introduced by formaldehyde. On the other hand, in an alkaline environment (pH 8.0), chelation of calcium is responsible for antigen retrieval and can be carried out with a chelator. Dixit et al. (1986) had also proposed earlier that the removal of calcium by chelation modifies the conformation of protein molecule as an unrolling or unraveling of the large domains, resulting in the exposition of epitopes necessary for the binding of certain monoclonal antibodies. A somewhat different interpretation of the relationship of aldehyde fixation with the masking of antigens with calcium-protein complexes is reported by Shi et al. (1999a). According to this point of view, although calcium-induced modification of the protein molecule does occur and can be demonstrated immunohistochemically, it is independent of formalin-induced crosslinking. Addition of calcium chloride can reduce or alter immunostaining, but it is not related to the pH of this solution. In conclusion, the effects of calcium bound to tissue are highly complex, for calciuminduced molecular modification may diminish antibody-antigen recognition or enhance this effect. It is known that the ability of some monoclonal antibodies to recognize their corresponding epitopes is calcium-dependent under certain conditions. The presence of citrate buffer is not necessary to restore antigenicity, provided an appropriate pH is present. The effect of bound calcium is not the only factor responsible for antigen masking. In addition, modification of a protein molecule also occurs due to crosslinking introduced by formalin fixation, causing antigen masking. Calcium binding to protein molecules influences the immunoreactivity of some epitopes, while others are not affected. On the other hand, heat-induced hydrolysis of protein crosslinks is the primary mechanism responsible for epitope unmasking. Possible mechanisms responsible for epitope retrieval by heat treatment are summarized below. In summary, reasoned arguments have been presented in support of several mechanisms responsible alone or in combination for antigen retrieval by heating; denaturation and hydrolysis, self-assembly of unfolded protein chains and the subsequent restoration of antigenic sites, chelation of calcium complexes, and unfolding of protein structure by metallic salts or urea solutions through dissociation of hydrogen bonds or through the loss of diffusable blocking proteins (Macintyre, 2001). In this respect, the role of residual paraffin in the sections is unclear.

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USE OF ETHYLENEDIAMINETETRAACETIC ACID (EDTA) FOR ANTIGEN RETRIEVAL
Epitope unmasking can be achieved by exposing sections to high temperature in combination with a calcium-chelating or precipitating agent such as EDTA or EGTA. In fact, maximum antigen retrieval induced by heat is obtained in the presence of EDTA. Such a treatment leads to the extraction of calcium ions tightly complexed to formaldehyde-fixed tissue sections. EDTA solution compared with other antigen retrieval fluids, including sodium citrate buffer, is more effective in certain cases in augmenting not only staining intensity but also the number of positively stained cells. It has been reported that EDTA solution, when combined with heating in a pressure cooker, is more effective than citrate buffer or Tris-HCl buffer in retrieving Ki-67 antigens in gastric and breast cancer tissues (Kim et al., 1999). The intensity of immunostaining of the sections treated with the EDTAheat combination depends on the pH of EDTA. Strong staining is achieved at pH 3 and at neutral to high pH levels, but the staining intensity decreases at pH 4 and 5. Another recent study also supports the mediation role of EDTA in unmasking antigens (Röcken and Roessner, 1999). In this study thin sections of aldehyde-fixed, Eponembedded human biopsy tissues were treated with 1 mM EDTA, using a heated water bath. This combined treatment resulted in excellent immunogold staining of amyloid (Fig. 6.4).

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Because heating in water alone did not improve immunostaining, it is concluded that epitope retrieval is mediated not only by heat and rehydration but also by the presence of a chelating agent such as EDTA. However, caution is warranted in using EDTA, which may adversely affect cell morphology because it is a strong oxidant.

ANTIGEN RETRIEVAL WITH HEAT TREATMENT
Theoretically, any method of heating should unmask epitopes. Although most antigens can be detected after heat treatment, some may be destroyed, and others may remain masked. The temperature, changes in temperature during heating, and the duration of heating critically influence antigen retrieval. Different tissues, fixatives, and durations of fixation require specific temperatures and durations of heating. Therefore, pilot studies should be carried out to determine optimal heating conditions. Similar staining intensities are achieved by the following heating conditions irrespective of the heating method used: 100°C for l0min, 90°C for 30min, 80°C for 50 min, and 70°C for 10hr (Shi et al., 1995a). Heating at 100°C for l0min (two cycles of 5min each) is recommended for retrieving most antigens, except those damaged by high temperature. In the latter case, lower temperatures for extended durations can be used. Overfixed tissues require high temperatures and/or extended durations of heating. In some cases, durations of heating longer than 10 min on a full-power setting may cause background staining. Repeating the boiling cycles is more effective than extending the boiling duration. This can be accomplished by removing the slide jar from the microwave oven after each run and placing the slides in a new jar containing the fresh retrieval fluid at room temperature, followed by again placing the jar in the oven. Compared with high-power microwave outputs, medium wattage (e.g., 450 W) may yield better sensitivity, probably due to optimal thermal effects and hence optimal oscillation of dipolar molecules.

ADVANTAGES OF HEATING
The recognition of many types of antigens by antibodies is facilitated using hightemperatures. Theoretically, high-temperature heating disrupts protein crosslinks introduced by formaldehyde, causes peptide cleavage, and alters protein tertiary structure, resulting in the exposure of masked epitopes for immunostaining. The retrieval of some types of antigens can be accomplished only by heating. Heating allows the use of antibodies that heretofore could not be employed on sections of tissues fixed with formaldehyde and embedded in paraffin. The heat-induced antigen retrieval procedure lowers the detection threshold for the antigen and improves signal-to-noise ratios; this is true for both monoclonal and polyclonal antibodies. In addition, the development of heating methods permits abandoning the use of frozen tissues, which are difficult to process and study. Another advantage of heating is that it allows the detection of antigens resistant to proteolytic enzyme digestion and retrieves antigens on sections of tissues left in formalin for prolonged durations. Generally, heat treatment is superior to enzyme digestion for antigen retrieval. For example, when using polyclonal anti-kappa and anti-lambda antibodies (1:500),

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immunoglobulin light-chain immunostaining was better after microwave heating than after trypsin digestion (Ashton-Key et al., 1996). Deparaffmized sections were heated on full power in a 750 W microwave oven for 22min, while others were treated with 0.1% trypsin for 10 min at 37°C. Similarly, in human biopsy tonsil tissue stains better after microwave heating than after trypsin treatment (Fig. 6.5). If the retrieval of an antigen type is adversely affected by heating, protease digestion of sections is the optimal pretreatment. However, the preservation of cell morphology is generally better in heat-treated than in enzyme-treated sections, especially when extended durations of digestion are employed.

HEATING METHODS
Different heating systems, such as microwave ovens (Shi et al., 1991), pressure cookers (Norton et al., 1994; Miller and Estran, 1995), microwave heating–pressure cookers (Taylor et al., 1995), autoclaves (Bankfalvi et al., 1994), steamers (Taylor et al., 1995), water baths (Kawai et al., 1994), and electric hot plates (von Wasielewski et al., 1994), in combination with antigen epitope retrieval fluids, have been used with various degrees of success. Although each of the methods has minor advantages and limitations, they yield a fairly similar degree of antigen retrieval when appropriate heating conditions are provided. All the processing conditions must be adjusted for a specific study. Such conditions may differ from those most widely cited in the literature or recommended by the manufacturers. The choice of the heating method also depends on equipment availability. A recent comparative study of the following five heating methods using 21 antibodies also demonstrated that they produce similar intensities of immunostaining of retrieved antigens provided the heating durations are adjusted appropriately (Taylor et al., 1996b). However, heating methods Nos. 2, 3, and 4 (given below) yield better results. Advantages and minor limitations of the heating methods are listed below. 1. Microwave heating for 10 min, carried out in a standard, simple, inexpensive, and widely available microwave oven, is the fastest procedure. Total time required (including set up of preheating, actual retrieval process, and cool down) is 25 min. A limitation is possible boiling over, resulting in the loss of antigen retrieval fluid. Consequently the level of the fluid must be checked every 5 min. If necessary, more fluid can be added after the first 5 min to avoid drying the tissue sections. If more fluid is needed, this is the result of boiling over, not evaporation. To catch any boiled-over fluid, the slide jar should be placed within a larger jar which contains deionized water. In addition, the presence of hot or cold spots in the microwave oven is not uncommon when several isolated jars containing the slides are placed at random in the oven, a practice that leads to reduced reproducibility. This method is also difficult to standardize. The microwave oven (900 W, 2,450 MHz) is set at maximum power for two cycles of 5 min each.

Step-by-Step Protocol
Determine the optimal pH of antigen retrieval solution for each antigen. Citrate buffer (0.01 M) adjusted to pH 6.0 with HC1 is used widely. Determine the desired temperature based on the type of tissue and antigen under study. For fatty tissues, 90°C is recommended; adjust the duration of heating accordingly. Place slides in plastic Coplin jars containing the

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antigen retrieval solution in the center of the rotary plate in the microwave oven to ensure uniform heating of slides. Cover the jars with loose-fitting caps. Turn on the microwave oven and check the temperature of the retrieval solution with a temperature probe. Use a maximum power setting of 7–10. Start timing the antigen retrieval duration when the retrieval solution begins to boil. As an average, a total retrieval duration of 10 min, divided into two 5-min cycles with an interval of 1 min between cycles to check the solution level in the jars, is recommended. If needed, fresh retrieval solution from an adjacent jar can be added to the jars containing the slides. Alternatively, distilled water can be used to replenish the retrieval solution. The objective is to keep the slides fully immersed in the solution before restarting the oven. Alternatively, the duration can be based on previously determined time for sections known to contain the antigen under study. Remove jars from the oven, and cool the sections for 20 min at room temperature. The slides are ready for immunostaining after being rinsed in 0.5 M PBS (pH 7.4) for 5 min. Do not reuse the antigen retrieval solution. Some of the above-mentioned steps are automatically controlled by the H2550 Laboratory Microwave Processor. 2. Microwave heating for 20 min is the same as Method 1 except that the heating is employed four times for 5 min each. Total time required is 35 min. Improved immunostaining of many types of antigens can be achieved by extending the heating time. The procedure requires attention for 20 min to check the fluid level, and occurrence of hot or cold spots may complicate the procedure. 3. Pressure cooking. Although microwave heating is widely used for antigen retrieval, this system does not raise the temperature of an aqueous buffer above 100°C, even though this temperature is reached rapidly. In contrast, an advantage of the pressure cooker is that, if required, temperatures of 115°C or higher (superheating) can be achieved. Other advantages of heating in a pressure cooker include short duration of heating, better reproducibility of results with large batches of slides, the ability to use metal slide racks, and economy of time and equipment cost (Norton et al., 1994).

Step-by-Step Protocol
Fill to approximately one-third capacity of a domestic pressure cooker (103kPa/15 psi) with 0.1 mM citrate buffer (pH 6.0). Bring the buffer to a boil using an electric hot plate, without sealing the lid. Quickly place metal racks containing rehydrated sectionmounted glass slides into boiling retrieval buffer, and seal the pressure cooker. Bring the cooker to full pressure. Start timing when the pressure indicator valve reaches the maximum (~4 min). The optimal duration of pressurized boiling is 1–2 min. Depressurize the cooker and cool it under running tap water. Remove the lid, and add cold tap water to replace the hot retrieval buffer. A duration of 15–20 min is required to cool the cooker. Wash the slides in several changes of 0.05 M PBS (pH 7.4) prior to immunostaining. At no time during this processing are the slides allowed to dry out. The pressurized boiling (120–122°C) longer than ~2min will progressively degrade the cell morphology. 4. Pressure Cooker–Microwave heating The pressure cooker–microwave heating method is simpler than the autoclave procedure and more efficient than microwave heating alone. The pressure cooker does not require checking the level of the antigen retrieval solution during heating in the microwave oven, and a large number of slides can be loaded simultaneously. In addition, the pressure

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cooker does not develop cold spots because it is a larger container. The limitation is that a slightly longer duration (~45 min) is required than that for microwave heating alone.

Step-by-Step Protocol
Place slides in three plastic staining jars, each containing 24 slides and antigen retrieval solution, and transfer them into a plastic pressure cooker (Nordieware, Minneapolis, MN) filled with 600 ml of distilled water; this amount of water is one-half the capacity of the cooker. Make sure that the jars stand stably in the water (Taylor et al., 1996b). Transfer the pressure cooker into a microwave oven (model R-4A46) which is equipped to switch one power level setting to another automatically. Place the cooker in the center of the microwave oven. Set the oven at maximum power (900 W, 2,450 MHz) for 15 min to boil the water, then switched to a 40% power setting for an additional 15 min to maintain mild boiling (simmering). Remove the cooker from the oven, and allow it to cool for 15 min. 5. Autoclave heating, like pressure cooking, provides superheating at temperatures higher than 100°C. Hydrated autoclaving eliminates the need to adjust the volume of the antigen retrieval solution. Other advantages include the use of larger volumes of the retrieval solution, which gives a uniform heating pattern and allows the heating of a large number of slides in a single batch. In this method high-intensity immunostaining is achieved, and the cold spots are absent. Hydrated autoclaving of slides, even in deionized water, is thought to be more effective than either microwave or water bath heating (Shin et al., 1991). It is known that heat denaturation of antigens is effective when the protein is hydrated, whereas dehydrated protein is extremely resistant to heat denaturation. Minor limitations are that the autoclave is expensive and may not be available in some small laboratories. Also, the total time required to complete heating is ~45 min. Care should be taken in handling owing to the pressure in the autoclave. The results of autoclave antigen retrieval are shown in Figure 6.6 (Plate 3C, D, E).

Step-by-Step Protocol
Place slides in Coplin jars containing antigen retrieval solution that has been previously heated at 80°C. Set the jars in the center of a stainless steel autoclave equipped with a 1,850 W heating filament. Tightly close the door of the autoclave as required by the instructions, and heat at 120°C for 10 min at 15 psi. Cool down with running tap water for 20–30 min, then rinse the sections with 0.05 M PBS at room temperature and immunostain. 6. Steam heating for 20 min over boiling water. Total time required is 35 min. This method has the advantage that loading and unloading of slides into various carriers for automated use is not required. It needs relatively small amounts of the antigen retrieval fluid, does not have cold spots, and is inexpensive. This approach is well suited to process a large number of slides simultaneously and thus saves considerable amount of time. Although originally designed for autostaining, it can be used manually. A minor limitation is that preheated steam is needed. Slides are set into the TechMate slide holder (Biotek, Santa Barbara, CA), with antigen retrieval fluid in the capillary gap, and are heated by steaming over boiling water. In contrast to microwave heating, steam treatment heats slides slowly to a uniform temperature. This avoids boiling the antigen retrieval fluid and minimizes section detachment from slides. Steam heat used in combination with EDTA and protease digestion has been

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reported to be superior to other antigen retrieval techniques for the immunostaining of cytokeratin (using antikeratin antibody) in select cases of prostatic carcinoma (Iczkowski et al., 1999). This approach minimizes the diagnostic ambiguity often encountered when using this antibody. Figure 8.8 shows clearly the superiority of the steamEDTA-protease method over protease treatment alone in detecting acinus with high-grade prostatic intraepithelial neoplasia. However, for a similar study, the hot plate method is preferred (see below). 7. Hot plate heating is the simplest and fastest heating method to retrieve antigens in sections of formalin-fixed and paraffin-embedded tissues. Certain antigens are optimally retrieved with the hot plate heating procedure than with enzyme digestion or microwave heating methods. The hot plate heating method is also most effective in retrieving certain antigens in tissues fixed with formalin for as long as 1 month. The retrieval of basal cell–specific, anti-high-molecular-weight cytokeratin (HMCK) in sections of radical prostatectomy specimens fixed in formalin and embedded in paraffin has been accomplished by using the hot plate method; monoclonal antibody clone raised against human stratum corneum was used in this study (Varma et al., 1999).
Step-by-Step Protocol

Sections mounted on a glass slide are placed in a beaker containing 1,000 ml of 0.2 M citrate buffer (pH 6.0) and heated on a hot plate (Corning, Utica, NY) for l0min at 100°C, and then allowed to cool at room temperature for 20min. 8. Equally good results, if not better in some cases, can be obtained with overnight treatment of tissue sections in Tris buffer (pH 9.0) in a conventional oven at 70–80°C

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(Koopal et al., 1998). This method has been successfully used for retrieving antigens such as estrogen and bcl-2 in cervix tissue and lymph node, respectively. Hot oven heating can also be tried in a humidified chamber. 9. Another antigen retrieval method is hot water bath heating at 90°C for 120min. Total time required is about It is simple and inexpensive, processes a large number of slides each time, and does not require replenishment of the antigen retrieval fluid. The method has been successfully used for the immunostaining of p53 and proliferating cell nuclear antigens (PCNA) (Kawai et al., 1994). For p53 and PCNA retrieval, 0.01 M PBS (pH 7.2) and 0.01 M citrate buffer (pH 6.0), respectively, are recommended (Fig. 6.7). The use of this method is limited since it takes much longer time to complete. Hot oven heating in a humidified chamber can also be tried.

Mechanism of Epitope Retrieval by Microwave Heating
The effects of microwave heating on the tissue sections that result in epitope retrieval are exceedingly complex. A full understanding of the actions of microwaves at the molecular level to facilitate epitope retrieval is lacking. At least two mechanisms need to be considered: heat and kinetic energy of the oscillating electromagnetic field. Both possibilities are discussed below. The most commonly accepted point of view is that heat is responsible for unmasking the epitopes. In fact, Battifora (1996) has introduced the phrase heat-induced epitope retrieval (HIER). Heating at 100°C is a powerful treatment that can unmask hidden, buried, or crosslinked epitopes. Heat can be provided not only by a microwave oven, but also by an autoclave, a pressure cooker, steam, or a hot plate. A consensus on which method of heating is most effective in the retrieval of all types of epitopes is lacking. Therefore, some

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factor or factors in addition to heat also become relevant. It is also known that treatments other than heat can also unmask epitopes. Such treatments include enzyme digestion and exposure to detergents. The aforementioned observations indicate that heat is not the only mechanism responsible for epitope retrieval. Therefore, the question arises, is the heat in the microwave oven the only factor or the primary factor that facilitates epitope retrieval? Is it possible that in addition to heat, kinetic energy plays a part in epitope unmasking (personal communication, A. S.-Y. Leong)? It is known that the microwave electromagnetic field causes polar molecules in the tissue to oscillate at a rate of 2.45 billion cycles per second, enough to disrupt protein crosslinking and unmask hidden epitopes. In addition, the fact that ultrasound treatment, which generates heat for an exceedingly short duration, also unmasks epitopes, suggests that factors other than heat may also be important in explaining the phenomenon of epitope retrieval in a microwave oven. The following explanation may further understanding of the release of thermal energy and heat in a microwave oven. Microwave energy is a nonionizing radiation (frequency, 300–300,000 MHz) that causes molecular motion by migration of ions and rotation of dipoles. Dipole rotation refers to the alignment, due to the electric field, of molecules that have either permanent or induced dipole moments in both the solvent and specimens. As the field intensity decreases, thermal disorder is restored, which results in thermal energy being released. At 2,450 MHz (the frequency used in commercial systems), the alignment of the molecules followed by return to disorder occurs times per second, resulting in rapid heating. However, the absorption of microwave energy and its release as heat are strongly dependent on the relative dielectric constant (relative permittivity) and the dipolar status of the medium. The relative permittivity is the following ratio: material dielectric constant: vaccum dielectric constant. The greater the relative dielectric constant, the more thermal energy released, and the more rapid the heating for a given frequency (Camel, 2001). Due to the particular effects of the microwaves on matter (namely dipole rotation and ionic conductance), heating of the section, including its core, occurs instantaneously, resulting in rapid breakdown of protein crosslinkages. Furthermore, the extraction and recovery of a solute from a solid matrix with microwave heating is routinely obtained in the field of analytical chemistry (Camel, 2001). However, a definite, full explanation of the effects of microwave heating on the molecular aspect of antigen retrieval is awaited.

Duration of Microwave Heating
The duration of microwave heating to retrieve epitopes depends on the type of concentration of the aldehyde used for fixation, duration of fixation, and the temperature in the microwave oven. The higher the concentration of the fixative and the longer the duration of fixation, the higher the temperature and the longer the duration of microwave heating required for epitope retrieval. The oven temperature is controlled using the temperature probe of the oven and is checked with a thermometer. In a microwave oven with 720 W power, the boiling point for the epitope retrieval fluid in the Coplin jar is reached in 140–145 sec (Shi et al., 1994). The time it takes to reach a temperature of 55°C is ~76sec. At 720 W, 5–10 min heating time is recommended, which can be divided into two 5-min cycles with an interval of 1 min between cycles to check on the fluid level in

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plastic or glass jars. If necessary, more fluid at the same pH can be added after the first 5 min to avoid drying the tissue sections. The jars can be covered with perforated cling film to minimize evaporation. Alternatively, 1 hr at 55°C or 30–120 min at 90°C (not boiling) can be used but is not preferred. With long durations (24 hr–3 years) of fixation with formaldehyde, 20 min exposure to heating at 100°C is recommended (Fig. 6.8) (von Wasielewski et al., 1994). A thorough washing of slides after microwave heating in the presence of epitope retrieval fluid and before incubation is essential to avoid background staining.

Antigen Retrieval in a High-Pressure Microwave Oven
Although microwave heating, pressure cooking, wet autoclaving, and steaming of tissue sections yield satisfactory antigen retrieval results, comparative studies indicate that pressure cooking or pressure cooking in combination with microwave heating produces more uniform, efficient, consistent, and rapid immunostaining in some cases (Fig. 6.9). Pressure cooking with or without microwave heating provides temperatures higher than 100°C (superheating). Such temperatures can be obtained with the high-pressure microwave processor MicroMED URM (Sorisole, B G; Bergamo, Italy) (Suurmeijer and Boon, 1999). This apparatus provides controlled superheating under high pressure in the microwave processor. This processor has a maximal power output of 1,000W. The duration, temperature, and pressure can be adjusted with a touch screen personal computer. Microwave power and pressure are controlled through software. The pressure is regulated as a function of temperature, which facilitates heating of the antigen retrieval solution at a constant temperature higher than 100°C without bubbling. A glass dome designed to withstand pressure conditions rotates within the microwave cavity. The dome is provided with an automatic raising and lowering mechanism controlled by the personal computer. A fiberoptic sensor monitors the temperature of the antigen retrieval solution within the dome. The pressure in the glass dome is between 1,900 and 2,000 mbar. To obtain antigen retrieval, a plastic jar containing 250ml of 0.01 M citrate buffer (pH 6.0) is centrally placed in the dome within the microwave cavity. Different temperatures (ranging from 90–115°C), durations of heating (1–15 min), and pH values (2–10) can be tested to determine optimal parameters for retrieving a given antigen. For example, optimal immunostaining of Ki-67 antigen in malignant tumors using MIB-1 antibody was achieved at 115°C for 10 min at pH 6.0 (0.01 M sodium citrate buffer) (Suurmeijer and Boon, 1999). To my knowledge the use of this processor has not been reported by any other laboratory, perhaps because of its high price.

Antigen Retrieval at Low Temperature
Heating treatment is one of the most important factors influencing the effectiveness of antigen retrieval on tissue sections. The heating of sections of the formalin-fixed and paraffin-embedded tissues at a high temperature (boiling) for 10–20 min is extensively used for retrieving many types of antigens. A variation of this method consists of heating at high temperature, followed by heating at moderately low temperature.

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Although antigen retrieval at high temperature yields excellent results in terms of nuclear immunostaining, cell morphology can be severely damaged. Such damage is either ignored or not readily visible at the resolution provided by the light microscope. Electron microscopy clearly shows this damage. The use of high-temperature heating is especially undesirable when studying fatty and fibrous tissues such as breast, skin, and gastrointestinal

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tract; morphological preservation of these tissues can be difficult at high temperatures. Also, sections of these tissues may be dislodged from slides (coated or uncoated) at high temperature. In some cases, regional staining of the section may also result from high temperature heating. The above-mentioned problems can be generally circumvented by using moderately low temperatures (60–80°C) for antigen retrieval. Such temperatures are also useful for antigen retrieval on archival sections that have been stored for years at room temperature on coated or uncoated slides. Dislodging of archival sections from slides during antigen retrieval is also minimized at moderately low temperatures. According to Biddolph and Jones (1999), these problems can be minimized by using low-temperature heating (60°C) in conjunction with boric acid as the antigen retrieval fluid. Loss of archival sections is especially serious because of the nonavailability of tissue blocks or if all the tissue has been used. If high temperature must be used for archival sections, loss of sections may be minimized by using an adhesive overlay on the sections before heating (Pateraki and Kontogeorgos, 1997). This approach requires further testing. Equal staining intensity is usually achieved at 80°C or at higher temperatures. Antigen retrieval can be obtained at 80°C in 10 mM citrate buffer using a water bath. A minor disadvantage to using moderately low temperature for antigen retrieval is the requirement of longer heating durations. The lower the temperature, the longer the duration of heating. As an average, heating at 80°C for 2hr is recommended. A wide range of heating durations at moderate temperature has been used in the published literature, which are listed below. Overnight heating (~ 15 hr) at 60°C has been used for retrieving muscle actin (HHF 35) and smooth muscle actin (CCG 7) (Igarashi et al., 1994). The same duration of heating but at 80°C was used in a conventional oven for retrieving estrogen receptor in the cervix tissue; Tris buffer (pH 9.0) was used as the antigen retrieval fluid (Koopal et al., 1998). Kawai et al. (1994) have reported that simple heating in a hot water bath at 90°C for 2hr was very effective for retrieving PCNA and p53 antigens (see Fig. 6.4). In this study, overnight heating at 60°C, using PBS or citrate buffer, also yielded good results. Heating at 90°C in a microwave oven for 15 min was also used for retrieving myosin heavy chain, using TUF antigen retrieval fluid; boiling was not allowed (Carson et al., 1998). Man and Tavassoli (1996) found that overnight heating at 70–80°C produced excellent staining of a number of antigens, including ER, PR, p53, and Ki-67. Moderately low temperature in conjunction with enzyme digestion has also been recently used for antigen retrieval; a few examples are cited below. The retrieval of Ki-67 antigen in surgical breast biopsy specimens has been achieved by treating the sections with 0.1% trypsin for 15 min at 37°C, followed by heating in 10 mM citrate buffer (pH 6.0) at 80°C for 2 hr in a water bath (Elias et al., 1999). In another study sections of breast cancer tissue were pretreated with 0.1% trypsin in PBS (preheated to 37°C) for 15 min, rinsed in deionized water, and then heated in l0 mM citrate buffer (pH 6.0) (preheated) in a water bath for 2hr for improved retrieval of estrogen receptor and Ki-67 antigen (Frost et al., 2000). A disadvantage of this combined treatment is the focal and sporadic digested appearance in the sections. These areas can be identified by the presence of inadequately stained nuclei by hemotoxylin. Moderate heating with or without enzymatic pretreatment is not the optimal method of immunodetection of all antigens. For example, more intense staining of antiapoptotic

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protein bcl-2 and progesterone in breast carcinoma is provided by heating in a microwave oven at full power than by moderate heating, although section loss is not uncommon with full-power heating. It is deduced from these and other studies that optimal retrieval of each antigen or antigen-antibody complex along with the tissue type requires specific intensity of temperature and its duration, and that the source of heat is not very important. Unfortunately, a universal temperature for retrieval of all antigen types in various tissues remains elusive.

Use of Heat for Staining
Microwave heating accelerates the process of staining for light and electron microscopy, although this advantage is more useful for light microscopy because conventional staining is quite rapid for electron microscopy. Under microwave heating, the charged dye ions as well as the polar molecules and ions of the solvent, including water, are excited (Kok and Boon, 1990). The molecular movement generated by microwave heating may accelerate chemical reactions up to 1,200 times. The result is that heating speeds up diffusion of stains into the thick-tissue sections and their subsequent reaction and binding with the substrate. Generally, compared with conventional staining, staining in the microwave oven requires a much shorter staining duration and results in more intense staining, better contrast, and less nonspecific staining. In fact, the hours required for many conventional staining methods for light microscopy can be shortened into minutes. Several examples are listed below. One example is the Grimelius method for staining neuroendocrine granules in various tissues and tumors for light microscopy. The conventional procedure is completed in 3 hr, whereas the microwave method is accomplished in 3 min (Hopwood, 1992). Also, staining of melanin can be carried out with colloidal silver nitrate in 45 sec under microwave heating (Leong and Gilham, 1989a). Microwave heating is also effective in reducing the tissue staining time from 70min to 15min for localizing acid and neutral mucins with a modification of alcian blue periodic acid–Schiff stain (Matthews and Kelly, 1989). Microwave heat–stimulated staining of the brain tissue with the Rio-Hortego silver impregnation technique can be completed within 24 hr instead of the 7 days required by the conventional method (Marani et al., 1987). Satisfactory silver impregnation of cell bodies, axons and their terminals, and dendrites and their spines is obtained. Another example is the application of the Jones-Marres silver method for rapid staining of fungi in the brain tissue of immunocompromised patients (Boon et al., 1998). This procedure can be carried out using the MicroMED BASIC microwave lab station (Milestone, s.r.l., 24010 Sorisole, Italy). The lab station has software for reliable control of power, time, and temperature using infrared temperature control for no-touch temperature determination. It also has a 360-degree rotation carousel (no hot spots) and produces printouts of the temperature and power levels used during various microwave steps. Microwave heat can also be applied for rapid staining of frozen sections. Frozensection diagnosis plays an important role in the evaluation of the operability of the patient and in the examination of resection margins. The preparation for diagnosis, for example of signet-ring cell carcinoma in the peritoneum, can be accomplished in as brief a time as 30 sec by the modified periodic acid–Schiff’s (PAS) reaction facilitated by microwave heating

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(Dworak and Wittekind, 1992). This protocol can demonstrate even a small number of mucincontaining tumor cells surrounded by fibrous tissue in frozen sections (Fig. 6.10/Plate 3F). Microwave heat is also effective in staining SDS-polyacrylamide gels with Coomassie blue for visualizing as little as 5 ng of protein against a light blue background of the gel (Wong et al., 2000). This protocol is one of the most powerful methods in molecular biology for visualizing proteins. Rapid staining of frozen sections of human brain tissue that has been stored in 10% formalin (4% formaldehyde) for up to 10 years has also been reported (Feirabend and Ploeger, 1991). In this study, rapid staining was obtained in the microwave oven by using classic neuroanatomical staining methods such as Klüver-Barrera stain; originally Luxol fast blue step required up to 24 hr, whereas in the microwave oven this step needed only 15–60 min. Another application of microwave heating is rapid staining of plant tissues with dyes. For example, Safranin O can stain plant tissues in 45 min at 60°C in a microwave oven instead of the conventional 48 hr at room temperature (Schichnes et al., 1999). Microwave heat–assisted rapid fixation and double staining of the mouse fetal skeleton has also been carried out (Ilgaz et al., 1998). The staining was accomplished with a mixture of alcian blue and alizarin red S in 23 min in the microwave oven instead of 4 days at room temperature. The cartilage and bone are stained distinctly. Most staining methods require optimal temperatures and durations of staining. The optimal temperatures for most nonmetallic stains is 55–60°C, while for metallic stains it is 75–80°C (Suurmeijer et al., 1990). Some specific examples are given below: the Romanowsky-Giemsa method and the alcian blue technique at 55°C (Horobin and Boon, 1988), the Southgate mucicarmine procedure at 60°C, the Grimelius protocol at 75°C, the Grocott, Jones, and Fontana-Masson methods at 80°C (Kok and Boon, 1990), and the gold chloride (0.02%) at 74–98°C (Noyan et al., 2000). Some dye solutions, such as oil red O, can be used at boiling temperature.

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Table 6.1 indicates microwave power levels, the stain used, and the required temperature. Table 6.2 shows significantly reduced duration of staining under microwave heating compared with conventional staining conditions. However, many brands of microwave ovens are in use, and all vary in their performance, even at the same power level. Therefore, optimal stain concentration, temperature of staining, and durations of staining and rinsing will have to be determined for each type of new study. Also, care is required in interpreting the staining results because high temperatures tend to produce staining artifacts. Autostainers for histochemistry are available from the following sources: Leica Autostainer XL, Leica Instruments GmbH, Nussloch, Germany; Oticmax Rapid Microwave Histoprocessor Inc., 160 Shelton Road, Monroe, CT 06468.

Rapid Immunostaining of Frozen Sections
Rapid immunohistochemical study of frozen sections is necessary for intraoperative diagnosis in some cases. Rapid immunostaining is also helpful in confirming or excluding tumor clearance in resection margins or in detecting micrometastases in sentinel lymph nodes in breast cancer patients. Two methods to immunostain frozen sections are the enhanced polymer one-step staining (EPOS) system and the EnVision system; both systems are detailed later.

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Using the horseradish peroxidase method and microwave heating, Ichihara et al. (1989) were also able to immunostain frozen sections for the intraoperative diagnosis of pancreatic cancer in 30min. Only four different antibodies were tested in this study. A shorter duration of 10min has also been used for immunostaining frozen sections with the EPOS system (Chilosi et al., 1994). The EPOS procedure is based on the chemical linking of primary antibodies and horseradish peroxidase to an inert polymer complex (dextran) (Bisgaad et al., 1993). This methodology has been employed for immunostaining of Ki-67, PCNA, cytokeratin, and leukocyte common antigens (Tsutsumi et al., 1995; Richter et al., 1999). The limitation of the standard EPOS system is that the primary antibodies are labeled and thus are commercially available only for limited range of antigens. In contrast to the EPOS system, a modification of the highly sensitive two-step irnmunohistochemical EnVision system allows the detection of a broad spectrum of antigens in frozen sections in less than 13 min (Kämmerer et al., 2001). In this study 38 out of 45 antibodies tested showed specific staining. In fact, the modified EnVision procedure allows the use of any suitable primary antibody, preferably monoclonal antibodies. Like the EPOS system, EnVision employs a dextran polymer coupled to horseradish peroxidase molecules for detection. No attempt was made to block endogenous peroxidase, nor was any antigen retrieval pretreatment used. Because of the very short incubation durations, a humid chamber is not required to avoid evaporation of immunoreagents. A minor disadvantage of the modified EnVision system is that it requires primary antibody concentrations four- to tenfold higher than those used in the conventional immunohistochemical procedures. Another limitation of this modified method is that only two slides with two sections each can be processed at any one time.

Enhanced Polymer One-Step Staining Procedure
Sections thick) of freshly frozen tissues are mounted on silane-coated slides and fixed with 4% buffered formaldehyde (pH 7.0) for 20 sec (Richter et al., 1999). The sections are rinsed in TBS (pH 7.4) for 15 sec, followed by incubation with EPOS antibody for 3 min at 37°C in an incubation chamber. They are rinsed twice for 15 sec each in TBS, and then developed with peroxidase-DAB detection kit (Dako) in a microwave oven (500 W) for ~ 1 min; during microwaving, the slides are cooled by a cold water bath (Werner et al., 1991). After being rinsed in tap water, the sections are counterstained with hematoxylin for 10sec. They are rinsed in tap water and cover-slipped.

Modified EnVision Procedure
Tissue specimens are snap-frozen in liquid nitrogen for 30 sec immediately after removal and then transferred to a cryostat (Kammerer et al., 2001). Serial frozen sections of thickness are cut and placed on silane-coated slides. They are air-dried for 30 sec, fixed in acetone for 1 min at room temperature (22°C), and air-dried at 22°C (Fig. 6.11). The sections are incubated with primary antibody in the antibody diluent (Dako) for 3 min by placing the slide horizontally on a hot plate at 37°C. (All incubation steps are carried out by placing the slide horizontally on the hot plate at 37°C.) Following a brief rinse in TBS, the sections are incubated with the goat-anti-mouse EnVision-HRP-enzyme conjugate for 3min at 37°C.

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The sections are rinsed in TBS, and then exposed to DAB+ chromogen (Dako) for a few minutes as the substrate for the EnVision-HRP-enzyme. The sections are washed by shaking the slide rapidly under tap water for 10 sec. The excess fluid is removed from the slide with a paper towel. The slide is dipped in distilled water, counterstained with Meyer’s hematoxylin for 15 sec, and then rinsed in hot tap water at 42°C for 30 sec.

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Hazards and Precautions in the Use of Microwave Ovens
The causes of most hazards encountered in using a microwave oven are straightforward and can be avoided by taking necessary precautions. Higher-power settings and longer durations of heating than optimal for a given study should be avoided. Because overheating is not uncommon, the time setting should be checked. The fluid contents of the container heat faster than the container. In fact, the fluid contents of the container heat so fast that the container can still be cool (Marani, 1998). Even after the container has been removed from the oven, it will become hotter for a period of time. Changes in the size, shape, and nature of the container and its position in the microwave oven significantly change the temperature of the container fluid. Furthermore, changes of these factors will change the temperature of the container fluid even if the volume of the container contents remains unchanged. Overheating the microwave oven tends to result in boiling or excessively rapid evaporation of fluids such as ethanol used for dehydration, formaldehyde employed for fixation, and the antigen retrieval fluid. As a result, flammable and/or toxic materials are released in the microwave oven. Even without overheating, vapors are produced because containers are kept open in the oven to prevent pressurization. Transparent microwave containers should be used, fluid volumes should be ~100ml. Microwave ovens with attached efficient extractor fans are commercially available, as are microwave ovens with temperature probes. To avoid possible exposure to toxic vapors, the face should be turned away when the oven door is opened (Horobin and Fleming, 1990). The oven door should not be opened or closed to turn the microwave power on and off. When using Pelco 3440 MAX laboratory microwave oven (Ted Pella, Redding, CA), areas of high microwave flux should be checked, using a Pelco 3,614 microwave bulb array (Ted Pella) (Fig. 6.12). Specimens should not be placed in areas indicated by illuminated bulbs. Vials containing the specimens should be placed in a water bath (50 ml) that has been preheated to the required temperature. The temperature should be regulated by placing a microwave temperature probe into a vial of the same solution that is present in the specimen vial. The built-in temperature probe displays the temperature on the oven front panel. The wire that attaches the probe to the oven should be submerged in the water to decrease the antennae effect (Schichnes et al., 1999). An additional 400ml of static water load should be placed in the oven at an optimal position determined with the microwave bulb array. This water is changed between every step. Pelco BioWave microsystem is the latest advancement in microwave heating technology. It is equipped with vacuum cycling (down to 1 torr), variable wattages, and a precision temperature probe; it can accommodate Pelco coldspot connected to the Pelco load cooler and thus eliminate hot and cold spots during processing. The system can be used for both light and electron microscopy. The vacuum chamber is most helpful during fixation and infiltration of tissue specimens. The following specific steps must be taken while using a microwave oven for antigen retrieval (Marani, 1998).
1. Test microwave leakage with a microwave detector with a low sensitivity range. 2. Place the oven in an efficient fumehood. 3. Wear gloves while using your hands inside an oven.

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4. Predetermine the maximum power level of the oven to be used. 5. Do not place a container with a closed lid in the oven. 6. Do not use high temperature settings unless absolutely necessary. 7. Predetermine the heating time, and check the time setting. 8. Check the actual temperature attained by the specimen. 9. Predetermine the number of specimens to be heated. 10. Predetermine the exact position of the specimen in the oven during heating. 11. Predetermine the amount of a water load and its place in the oven. 12. Find out the extent of hot spots in the oven. 13. Use Teflon containers with thick walls in the oven. Plastic containers can also be used. 14. Do not use metals or foils in the oven. 15. Contrary to some published reports, pencil-written materials can be used in the oven.

Limitations of Microwave Heating
In spite of the overwhelming advantages of microwave heating, some real and possible limitations are described. Background staining may occur with some antibodies, particularly when the heating is prolonged. This problem can be avoided by determining the optimal temperature and time of heating by trial and error. Although antigen specificity

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for the monoclonal antibody is maintained after microwave treatment, the possibility of altered immunostaining should not be disregarded whenever new or previously untested antibodies are used. The results of such studies should be compared with those obtained using frozen section immunohistochemistry. In some cases, microwave heating may damage nuclear morphological details, including mitotic figures. This problem may lead to difficulty in identifying cells accurately, which is important in diagnostic studies. Both mitotic figures and morphology, for example, are important in distinguishing a malignant lymphoid infiltrate within a mixed cell population (Hunt et al., 1996). In such studies, it is desirable to use a heating method other than microwaves and accept slightly lower immunostaining enhancement. Although the exact knowledge of molecular changes responsible for impairment of nuclear morphology caused by microwave heating is lacking, it may be possible that this treatment causes some structural damage to intracellular macromolecules, resulting in an increase in the number of osmotically active moieties within the nuclear compartment, thus attracting water and causing nuclear swelling (Hunt et al., 1996). Such swelling would blur mitotic figures, leading to a less accurate count of them. Some other limitations and their avoidance are described below. With violent boiling and extensive evaporation of the retrieval fluid in which the sections are immersed, the sections should be monitored to avoid drying and damage. To obviate this problem, microwave heating must be performed in repeated bursts; the plastic jars must be refilled following each cycle or a large reservoir of retrieval fluid or distilled water must be placed in the oven. To avoid inconsistent results, plastic jars containing the slides should always be placed every time in the same location in the microwave oven. The number of slides and jars should be constant every time a microwave oven is used, even when this entails inserting blank slides into the jar (Gown et al., 1993). Tissue sections should be placed toward one end of the slide (lower side of the slide while placing it in the jar) to ensure continuous immersion in the epitope retrieval fluid during microwave heating. Uneven distribution of microwaves within the oven results in hot and cold spots (see pages 102–103). This problem can be avoided by placing a 500-ml water load in the rear of the oven and by using a turntable during the process of heating (Panasonic model NN 5652, 800 W). Only a limited number of slides can be accommodated in the microwave oven. Tissue section detachment from the glass slide may occur during heating, especially with tissues containing prominent fibrous elements (Cuevas et al., 1994). If this problem is encountered, the surface of the slide can be made adhesive for sections by coating it with poly-L-lysine or 3-aminopropyl-triethoxysilane or, still better, by using electrically charged glass slides. Another problem is that microwave ovens have the inherent disadvantage of decreased power generation with use. Thus, no two ovens in use will have the same heating characteristics. This limitation is an obstacle in standardizing antigen unmasking methods. Microwave heating in some cases is not desirable. This method, for example, causes complete loss of estrogen receptor immunoreactivity, even when monoclonal antibody H222 is used (Gown et al., 1993; Leong, 1996). In such cases, alternate procedures, such as enzyme digestion alone or followed by microwave heating, can be used. Similarly, another steroid hormone receptor androgen shows stronger immunostaining with autoclaving than that using microwave heating (see Fig. 6.13) (Ehara et al., 1996). Another example is insulin, which shows diminished immunoreactivity after microwaving in citrate

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buffer (pH 6.0) (Tornehave et al., 2000). In such cases alternative antigen retrieval fluids and heating methods are required. Contrary to some reports, microwave heating in some cases does not abolish contaminating immunostaining during the consecutive detection of two or more types of antigens within the same section. This problem is especially common when double immunolabeling with antibodies of the same species and isotope is used. Recently it was demonstrated that microwave heating did not completely abolish contaminating staining when cytoplasmic and nuclear antigens in proliferating cells were labeled in cryostat and paraffin sections, with primary monoclonal antibodies from the same species and the same isotope being used (Bauer et al., 2001). However, such contaminating staining can be avoided in some cases with the use of microwave heating. Lan et al. (1995) have reported blocking of antibody cross-reactivity in multiple immunoenzyme staining and retrieving antigens with microwave heating. In summary, although the microwave heating method is highly effective for detecting a large number of tissue-bound antigens which otherwise may remain masked, primarily due to fixation with formaldehyde, certain antigens show reduced immunoreactivity following microwave heating. It should also be noted that epitope retrieval with microwave heating or other methods can unmask cross-reactivities that can be very difficult to deal with. Therefore, the use of retrieval methods for immunostaining creates the necessity for increased vigilance in the selection and interpretation of controls, both negative and positive.

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WET AUTOCLAVE METHOD
Wet (hydrated) autoclave pretreatment as an alternative to microwave heating was introduced by Shin et al. (1991) for unmasking tau protein on sections of brain tissue fixed with formalin and embedded in paraffin. This methodology was later modified by Bànkfalvi et al. (1994) for diagnostic antigen retrieval. The justification for employing autoclaving is that microwave heating is less desirable than equivalent autoclaving for the retrieval of certain antigen types. For example, microwave heating may damage nuclear morphological details, including mitotic figures. Some evidence indicates that cell morphology is preserved better with autoclaving than with microwaving. It has been suggested that autoclave heating does not cause loss of sections. The retrieval of certain types of epitopes requires temperatures higher than 100°C, which is provided by an autoclave (120°C). It is thought that damage by the super-high temperature to cell morphology is comparatively less in some cases. The super-high temperature has been reported to result in stronger immunostaining of steroid hormone receptors than with that obtained with microwave heating (100°C for two pulses of 5min each) (Fig. 6.13) (Ehara et al., 1996). Also, compared with microwaving, autoclaving produces superior immunostaining of progesterone (Mote et al., 1997). In addition, autoclaving yields better immunostaining of U2-OS cell nuclei for retinoblastoma susceptibility gene product (RB) compared with that achieved by using microwave heating (Tsuji et al., 1998). Furthermore, immunostaining of fibronectin epithelial nucleus in oral mucosa was not present on frozen sections, but such staining was achieved after autoclaving (Mighell et al., 1995). However in some cases, autoclaving is undesirable. This is exemplified by the abolition of immunostaining of calcineurin in the CNS neurons after autoclaving (Usuda et al., 1996). Autoclave pretreatment (100°C) may damage cell morphology of fatty tissues or tissues containing areas of fatty tissues (e.g., breast tissue). The extent of damage can be minimized by mounting the sections on protein-coated glass slides that have been allowed to dry for 48–72 hr before autoclaving (personal communication, 1999, K. W. Schmid). In addition to its effectiveness in antigen retrieval in the cases above, an autoclave has the advantage of accommodating a much larger number of slides than does a microwave oven. Several hundred slides can be simultaneously processed in an autoclave, eliminating possible immunostaining variations when small batches of slides are microwave-heated at different times. Moreover, antigen retrieval fluid is not lost during heating in an autoclave. In contrast, in a microwave oven the jar containing the slides must be refilled after each heating cycle. However, this tedious exercise can be avoided by using a large reservoir of fluid to minimize the possible deleterious effects of boiling or drying of the sections. A drawback of autoclaving is the high cost of the equipment. The use of a pressure cooker is a cheaper alternative for a smaller number of sections.

Procedure 1
Tissues are fixed with formalin for 18 hr to 4 weeks and then embedded in paraffin. Sections are mounted onto superfrost or poly-L-lysine–coated glass slides, dried in an oven for 1 hr at 60°C, and deparaffinized with three changes of xylene. This is followed by rehydration through a series of descending concentrations of ethanol. The slides are placed

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in plastic Coplin jars containing 0.01 M sodium citrate buffer (pH 6.0), which are heated in an autoclave for 5–10 min at 120°C. The slides are allowed to cool down to room temperature for 20–30 min and then briefly rinsed in 0.05 M Tris-HCl buffer (pH 7.4) or 0.1 M PBS. Blocking of endogenous peroxidase activity is accomplished by immersing the sections for 30 min in a solution of 0.3% in distilled water, and then rinsing in PBS. If needed, background staining can be blocked by treating the sections for 10–30 min at room temperature with normal serum from the species supplying the second antibody at a dilution of 1:5 to 1:20 in PBS. The sections are incubated overnight in a humidified chamber at 4°C in the primary antibody at an appropriate dilution. They are rinsed three times for 5 min each in PBS, further processed by using avidin-biotin complex (Vectastin, Vector Labs, Burlingame, CA), followed by DAB as the chromogen. Counterstaining of the nuclei is accomplished with hematoxylin or methyl green. As a negative control, irrelevant antibody UPC10 (Cappell, Organon Teknika, West Chester, PA) can be used or primary antibody can be omitted. The sections are mounted in an appropriate mountant.

Procedure 2
The following hydrated autoclave method can be employed for immunohistochemical detection of molecules in both cultured cell and tissue specimens. The method was used, for example, to localize androgen receptor in cultured LNCaP cells (derived from prostatic carcinoma metastasized to lymph node) and biopsy specimens from patients with prostatic carcinoma (Ehara et al., 1996). After being removed from the culture medium, the cells on plastic cover slips are fixed with 10% formalin for 10 min at 20°C. Tissue specimens are fixed for 1–2 days and embedded in paraffin. Sections are cut, mounted on glass slides, and heated in an oven for 1 hr at 42°C to promote adherence to the slide. After deparaffinizing and rehydration, the sections are subjected to epitope retrieval treatment as follows. The slides are placed in metal slide racks and immersed in a beaker filled with 0.01 M citrate buffer (pH 6.0). The beaker is loosely covered with a sheet of aluminum foil and autoclaved for 15 min at 120°C. After cooling to room temperature, the autoclave lid is taken off. The sections are treated with 3% hydrogen peroxide in methanol for 15 min to block endogenous peroxidase activity. As a blocking solution, 10% normal goat serum is used for 10 min. The sections are reacted with the primary antibody at an appropriate dilution at 4°C in a moist chamber. After being washed with 0.075% Brij 35 (Sigma Chemical Co., St. Louis, MO) in PBS three times, the sections are treated with an appropriately prediluted antibody for ~10 min in a moist chamber. After washing, the sections are reacted with the prediluted HRP-labeled streptavidin for ~5 min. The sections are washed, and the HRP site is visualized with DAB, hydrogen peroxide, cobalt, and nickel, without counterstaining. As a negative control, the sections are reacted with normal mouse serum, normal IgG, or normal rabbit serum in place of the specific antibodies after autoclaving (see Fig. 6.13).

ULTRASOUND TREATMENT
Ultrasound (sonication) converts AC line voltage to 20-kHz high-frequency electrical energy, which is fed to a converter where, in turn, it is converted to mechanical vibrations.

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The main part of the converter is a lead zirconate titanate electrostrictive element that expands and contracts when subjected to alternating voltage (Portiansky and Gimeno, 1996). The converter vibrates in a longitudinal direction and conveys this motion to the horn tip immersed in the solution, resulting in the implosion of microscopic cavities in the solution. The implosion causes the molecules in the solution to become exceedingly agitated. This phenomenon is explained below. Some information is available on the mechanisms responsible for the direct or indirect effects exerted by ultrasound on antigen retrieval. Considerable heat is generated during ultrasound exposure, but the heat dissipates very quickly. Very rapid heat loss has misled some workers to state that “ultrasound generates a mild increment in temperature” (Portiansky and Gimeno, 1996). Ultrasound waves consist of cycles of compression and expansion. Compression cycles exert a positive pressure on the liquid, pushing the molecules together, whereas expansion cycles exert a negative pressure, pulling the molecules away from each other. The tensile strength of solutions is reduced by gas trapped in the crevices of small solid particles in the solution. When a gas-filled crevice is exposed to a negative pressure cycle from a sound wave, the reduced pressure makes the gas in the crevice expand until a small bubble is released into the solution, initiating cavitation. A negative pressure of only a few atmospheres will form bubbles. The bubbles ( in diameter) implode violently in less than a microsecond, intensely heating their contents (Suslick, 1989). Thus, during the expansion cycle a sound wave of sufficient intensity can generate cavities in the solution. Ultrasound treatment causes enormous molecular agitation (turbulence), heat, and pressure of imploding cavities. Such agitation not only initiates but also accelerates both biochemical and physical reactions. In other words, effects of ultrasound involve processes that create, enlarge, and implode gaseous and vaporous cavities in a solution. The implosion of cavities also sends shock waves through the solution. This extreme condition generated by cavitation can induce reactivity between cellular proteins and the antigen retrieval solution (e.g., sodium citrate). Mechanical vibrations and high temperatures may extract tissue-bound calcium ions, accelerating the chelating effect of citrate. This suggestion is reinforced by the evidence that ultrasound hastens calcium chelation and bone decalcification (Thorpe et al., 1972; Page et al., 1990). Chelation of calcium may result in epitope retrieval (Morgan et al., 1994). It is known that ultrasound can break or disrupt cells and tissues. Mechanical vibrations generated by ultrasound can induce structural changes in the tissue sections, breaking the formalin-introduced protein crosslinks and thus facilitating the accessibility of antigens to antibodies. Ultrasound can also unfold or “crack” protein molecules into smaller fragments, exposing the epitopes. The effectiveness of ultrasound treatment in epitope retrieval has been compared with that achieved with microwave heating or pressure cooker alone (Portiansky and Gimeno, 1996). It was shown that ultrasound was more effective in immunostaining prostatic basal cell structural cytokeratins. The capability of microwave heating for epitope retrieval has also been compared with that of ultrasound in combination with microwave heating (Brynes et al., 1997). The latter approach resulted in stronger immunostaining with lower nonspecific background staining of cyclin Dl bcl-1 nucleoprotein in mantle cell lymphoma specimens. It should be noted that raising the temperature beyond a certain level in the presence or absence of ultrasound does not improve epitope retrieval and in addition results in excessive

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background staining. A limitation of ultrasound heating is that it is difficult to reproduce between laboratories, as the exact parameters regarding the intensity at the acoustic frequency are difficult to set precisely. The results of the studies carried out by Hammoud and Van Noorden (2000) using ultrasonication do not agree with those reported by Portiansky and Gimeno (1996). The former authors do not recommend using this technique in a routine histopathology laboratory. Miller et al. (2000) also report that the results obtained with ultrasonic treatment are inferior to those achieved with a pressure cooker. The discrepancy between the results obtained with these two teams and other workers may be due to the difference in the type of antigens ultrasonicated in various laboratories under different processing conditions. For example, Portiansky and Gimeno (1996) used an ultrasonic cell disrupter with an output of 40 W, whereas Hammoud and Van Noorden (2000) employed an ultrasonic cleaning bath having an output of 80 W. Also, various studies used tissues fixed for different durations. Tissue section adhesion to slides has also been reported to be a problem during ultrasonication. In spite of the lack of agreement on the usefulness of ultrasonic treatment, this method requires very short duration (~40 sec) for antigen retrieval. However, the usefulness of ultrasonic treatment requires additional substantiation.

Procedure
Tissues are fixed with 10% formalin for 7–10 days and embedded in paraffin (Portiansky and Gimeno, 1996). Sections about thick are mounted on glass slides coated with poly-L-lysine and deparaffinized with xylene. They are incubated with 0.03% methanolic hydrogen peroxide for 30 min to inhibit endogenous peroxidase activity. Following dehydration with graded ethanol, they are rinsed in deionized water and then in PBS. The glass slides containing these sections are vertically oriented in the lateral walls of a 75×95-mm glass dish and completely covered with 10 mM citrate buffer (pH 6). The tip of the cell is disrupted (Branson Ultrasonics model 250), set to continuous mode, and immersed 3 cm in the citrate buffer in the center of the dish. After incubation in the primary antibody (appropriately diluted), the avidin-biotin complex (ABC) is used as the detection system. In control sections, the primary antibody is replaced with normal mouse serum.

NONHEATING METHODS

Detergents
Antigen retrieval using heat-based methods is not being widely used for cell cultures and cryosections fixed with an aldehyde. The immunolabeling efficiency of such specimens can be improved by using a chemical antigen retrieval protocol. This protocol consists of permeabilizing the specimens with Triton X-100, followed by treating with sodium dodecyl sulfate (SDS). This permeabilization/denaturation treatment is applied after fixation and prior to incubation with the primary antibody. SDS is the most commonly used denaturing agent for gel electrophoresis. The application of SDS in epitope retrieval is based on the observation that after treatment with this reagent, protein bands appear in gel electrophoresis, but

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such results with similar proteins without SDS treatment using immunocytochemistry are not visible. Being a protein denaturant, SDS application may result in bands after staining with Coomassie blue, but it itself is not a stain. Therefore it should not be referred to as a positive or negative stain. SDS disrupts noncovalent interactions between subunits of a protein, so if a protein has two subunits, two bands will appear. In the absence of SDS, only one band will appear. This reagent and mercaptoethanol reduce protein subunits that are disulfide bonded. This property of SDS may be responsible for protein denaturation. It should be noted that SDS also permeabilizes cells for antibody access to intracellular epitopes. Triton X-100 and digitonin are also used to permeabilize the cell membrane allowing antibody penetration. Triton X-100 permeabilization of formaldehyde-fixed cells allows antibodies better access to their epitopes than does digitonin treatment. Digitonin or saponin binds to cholesterol within membranes, creating digitonin-cholesterol complexes and pores in the membrane. The pores are sufficiently large to allow antibody penetration. On the other hand, Triton X-100 is a stronger detergent and dissolves most of the membrane lipids. As a result this detergent increases the accessibility of antibodies to cell compartments that are not permeabilized with digitonin (Hannah et al., 1998). A limitation of Triton X-100 is that it may extract certain antigens even from fixed cells. Thus, false-negative staining of antigens, especially membrane antigens, of the cells treated with a strong detergent can occur because of antigen extraction. However, it should be noted that not all membranes of formaldehyde-fixed cells are impermeable to antibodies without permeabilization. The permeabilization/denaturation method has been successfully used for immunolabeling of in human neutrophils and MRC-5 cells (Robinson and Vandré, 2001). The method has also been effective in labeling MDCK cells in conjunction with indirect immunofluorescence (Brown et al., 1996). It should be noted, however, that some type of antigens remain masked, while other types may be adversely affected by SDS treatment. Still other antigens (e.g., aquaporins and brush border gp330) remain unaffected by SDS treatment (Brown et al., 1996). An example of an antigen whose staining is negatively affected by SDS treatment is in the Golgi complex; this occurs with the anti-AE1 anion exchanger antibody (Brown et al., 1996). Therefore, the usefulness of the SDS treatment should be assessed in each case. Caution is also required to prevent drying out of the specimens during incubation steps because they become hydrophobic with SDS treatment.

Procedures
Kidney tissue is fixed with paraformaldehyde-lysine-periodate by vascular perfusion (Brown et al., 1996). Tissue slices are further fixed overnight at 4°C with the same fixative and stored in PBS (pH 7.4) containing 0.02% sodium azide. They are placed in 30% sucrose in PBS for at least 1 hr, and then surrounded by a drop of Tissue-Tek embedding medium on a cryostat chuck before freezing by immersion in liquid nitrogen. Cryostat sections about thick are cut at a chamber temperature of –25°C, collected on Fisher Superfrost Plus charged slides, and stored at –20°C until use. The sections are brought to room temperature, and a wax pen (PAP pen, Kiyota International) is used to trace a hydrophobic circle around each section. They are rehydrated by immersion in PBS for 5 min; most of the PBS is removed from the slide with a tissue paper and the sections are then covered with drops of SDS solution (1% SDS in

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PBS). The drops are confined to areas where the sections are encompassed by the wax circles. After the slides have been treated horizontally for 5 min at room temperature, the slide is immersed in PBS in a Coplin jar to remove the SDS. The control slide not exposed to SDS is washed in a separate jar to avoid any contact with SDS. The slides are thoroughly washed three times for 5 min each with PBS, completely removing the SDS; otherwise, residual SDS will denature the antibodies subsequently applied to the sections. While the slide is horizontal, excess PBS from the areas outside the wax circles is removed. The sections become hydrophobic after SDS treatment, so care must be taken to prevent them from drying. The aliquot of primary antibody should be already in the pipette, so that it can be applied to the sections immediately after the residual PBS has been removed. The sections can be incubated in the primary antibody for 1–2 hr at room temperature, followed by two washes for 5 min each in high-salt PBS (containing 2.7% NaCl instead of 0.9% NaCl). This PBS minimizes nonspecific binding of antibodies to the tissue. After being washed for 5 min in normal PBS, the sections are incubated in the secondary antibody (goat antirabbit IgG conjugated to fluorescein isothiocyanate, FITC) for 1 hr. This is followed by washing in normal PBS, then mounting of sections in the medium of choice (Fig. 6.14). A second nonheating epitope retrieval method involves the use of sodium hydroxidemethanol solution. This solution was used successfully for epitope retrieval in sections of formalin-fixed, acid-decalcified human temporal bone embedded in celloidin (Shi et al., 1991). This solution is prepared by adding 50–100 g of NaOH to 500 ml of methanol in a brown bottle and mixing vigorously. The solution can be stored for 1–2 weeks at room temperature; it is also available commercially (BioGenex, San Ramon, CA). The clear, saturated solution is diluted 1:3 with methanol before use. A wider application of this solution is awaited. Another reagent used to unmask epitopes by denaturing antigens is guanidine hydrochloride (GdnHCl) which is freely soluble in water and alcohol; its

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aqueous solution has neutral pH. It was used for retrieving masked or hidden intracellular protein in scrapie-infected cultured cells (Taraboulus et al., 1990). A modified version of this protocol was employed for localizing different epitopes in BHK-21 cells fixed with paraformaldehyde containing small amounts of glutaraldehyde (Peränen et al., 1993). These epitopes were undetectable without denaturing the antigens with GdnHCl, using immunofluorescence microscopy. The advantage of denaturation of antigen complexes using GgnHCl is that it allows the use of glutaraldehyde, a more potent protein cross-linker, which better preserves protein cell structure. This means that low concentrations of this dialdehyde do not interfere with the epitope retrieval property of GgnHCl. The use of glutaraldehyde widens the applications of this denaturing agent. Both monoclonal and polyclonal antibodies can be used in conjunction with GgnHCl. Guanidine hydrochloride is especially useful with antibodies known to react only with denatured antigens. This reagent also permeabilizes cells. A limitation is that GgnHCl tends to eliminate the antigenicity of certain intracellular structures such as microtubules. However, microtubule loss can be prevented by using low concentrations of glutaraldehyde during fixation with paraformaldehyde (Peränen et al., 1993).

Proteolytic Enzyme Digestion
A variety of proteolytic predigestions have been employed for unmasking epitopes that had become inaccessible as a result of crosslinking during aldehyde fixation. The digestive treatments have been carried out most commonly with trypsin, pepsin, proteinase K, or pronase (their concentrations are given later) prior to immunostaining. Detailed comparative studies on the effects of these four enzymes on epitope unmasking demonstrate that while the results did not differ significantly among themselves, their effects did differ, depending on the tissue and the antibody used (Hazelbag et al., 1995). Other factors affecting such results include the duration of digestion, pH, temperature, and length of fixation. The mechanism responsible for antigen retrieval by enzymatic digestion is breakdown of protein crosslinks formed during formalin fixation. It is likely that enzyme treatment digests surface binding proteins, exposing the masked antigenic sites for antibody binding. This idea is supported by evidence that the duration of enzymatic digestion required for epitope retrieval is proportional to the length of formaldehyde fixation. It is also known that overdigestion leads to damage, not only to cell morphology but also to immunoreactivity. Enzymatic digestion is preferred over microwave heating for antigen retrieval in a few cases. Even multiple enzymatic digestion is required to retrieve certain antigens in a specific tissue. As an example, it has been reported that the monoclonal antibody RCC is most effective in the staining of clear cell carcinomas and papillary carcinomas in renal neoplasms when sections are pretreated with a three-step enzymatic digestion method: 0.12% trypsin in Tris-buffered saline (TBS), 0.01% pronase in TBS, and 0.1% pepsin in 0.1 N HC1. Results were inconsistent with heat-induced epitope retrieval techniques. However, trypsin is used most commonly, which catalyzes the hydrolysis of orginyl and lysyl peptide bonds. Trypsin usually is used at a concentration of 0.1% in 0.05 M Tris/HCl buffer (pH 7.8) containing 0.1% for 20–40min at 37°C. The addition of is essential

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for controlling digestion and reducing the production of occasional white flocculation (Macintyre, 2001). Only freshly prepared solutions of these enzymes should be used, as enzyme activity decreases with age. Also, these solutions should be prewarmed to the required temperature to ensure consistent results. Proteolysis does have certain limitations. Some antigens are susceptible to enzyme digestion. In some cases insufficient unmasking can result in poor or false-negative results, while excessive digestion may adversely affect cytomorphological features and cause increased background staining and detachment of tissue sections from the slide. It is known that proteolysis is a potent treatment. Because the cleavage of the protein molecule by proteolytic enzymes is mostly nonspecific, these reagents may alter the epitopes. In other words, peptide bond cleavage by these treatments is largely nonspecific. Therefore, these procedures are not the preferred treatments. Nevertheless, enzymatic digestion is useful for a limited panel of antibodies. If needed, enzymatic pretreatments can be applied preceded by microwave heating (Dookhan et al., 1993).

Procedure
Slides with tissue sections are treated with 0.1% trypsin solution containing 0.1% (pH 7.4) for 15 min at 37°C, with 0.4% pepsin solution containing 0.01 MHC1 for 20 min at 37°C, or with 0.025% pronase E solution containing 0.05 M Tris-HCl (pH 7.6) for 15 min at the same temperature (Hazelbag et al., 1995). These are average concentrations and durations, which should be adjusted according to the tissue and antigen type and the duration of fixation. Prolonged fixation requires longer proteolysis to unmask the epitopes. Excessive proteolysis results in decreased immunostaining. If loss of the sections during proteolysis is a problem, the slide can be coated with a 3% solution of casein white glue and dried overnight before the sections are placed on it.

Enzyme Digestion and Relatively Low Temperature (80°C)–Assisted Antigen Retrieval
High-temperature microwave heating is currently the most widely used antigen retrieval method. This approach has significantly improved the detection of a wide variety of antigens. In some studies low-temperature (80°C) antigen retrieval is more effective than that obtained with high temperature. This is exemplified by restoration of estrogen and progesterone immunoreactivity (Elias and Margiotta, 1997). Recently, it was reported that sequential use of trypsin digestion and low-temperature heating (80°C) was more effective than high-temperature retrieval of Ki-67 antigen in breast tumors, using MIB-1 antibody (Elias et al., 1999). Another reported advantage of the former approach is that it causes the least amount of section loss during heating; sections of tissues with a high fat content may be dislodged from the slide at a high temperature. Surgical breast biopsy specimens are first fixed with neutral buffered formalin (4%) for 4–6 hr, followed by zinc-formalin for 2 hr. Paraffin sections ( thick) are placed on silane-coated slides, dried on a slide warmer (60°C) for 1 hr and then in an oven (60°C) for an additional 1 hr. Deparaffinized sections are digested with 0.1% trypsin in PBS at 37°C for 15 min. The sections are placed in 10 mM citrate buffer (pH 6.0) and transferred into a water bath (80° or 90°C) for 2 hr. After a 20-min cooling period, the sections are rinsed

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with PBS and then incubated overnight at 4°C in the MIB-I antibody (Immunotech, Westbrook, ME), diluted 1:50 with PBS. An automatic immunostainer (Cadenza, Shandon Scientific Inc., Pittsburgh, PA) can be used to accomplish staining. The Supersensitive Streptavidin-AP detection kit (BioGenex, San Ramon, CA) is used according to manufacturer’s directions. The final color reaction is developed with a fast red substrate (BioGenex), followed by mild hematoxylin counterstaining to avoid masking weak immunostained nuclei. Slides are coverslipped with Crystal Mount (Biomedia Corporation, Foster City, CA).

COMPARISON OF ANTIGEN RETRIEVAL METHODS: A SUMMARY
The following recent comparative studies demonstrate that no single antigen retrieval method is optimal for all types of antigens.
1. Immunostaining of and isoforms of calcineurin in the human brain employing CAN-2 and CAN-3, respectively, was compared between microwave heating (in 10 mM sodium citrate at pH 6.0 for 10 min) and autoclaving at 120°C for 20 min (Usuda et al., 1996). The former approach was the most effective for intensification of the immunoreaction. 2. Compared to enzyme digestion methods, microwave heating demonstrated more intense immunoreactivity of estrogen and progesterone in breast cancer tissues fixed with methacarn (60% methanol, 30% chloroform, and 10% acetic acid) (Oyaizu et al., 1996). 3. Compared with trypsin digestion, microwave heating produced more consistent results and was effective over a greater range of fixation tissues in the case of immunoglobulin light chain in tonsil tissue (Ashton-Key et al., 1996). 4. Compared with pepsin predigestion, microwave heating markedly enhanced the staining of aberrant p53 antigen with Pab 1801-D07 antibody cocktail in paraffin or frozen sections in adenocarcinoma of the lung (Resnick et al., 1995). 5. Compared with microwave heating (three times for 5 min each at 100°C), hydrated autoclaving (5 min at 121°C) yielded stronger immunostaining of bcl-2 using bcl-2, 124 antibody (Umemura et al., 1995). 6. Compared with nonhydrated autoclaving, hydrated autoclaving produced stronger immunostaining of tau (a microtubule-associated protein) using anti-PHF/tau and antihuman tau (Shin et al., 1991). 7. Compared with microwave heating, heating on a hot plate yielded better immunostaining of IgG using antihuman IgG on epoxy thin sections for electron microscopy (Stirling and Graff, 1995). 8. Compared to microwave heating, superheating (120–122°C) for 1–2 minutes in a pressure cooker gave better immunostaining of IgD (Norton et al., 1994). 9. Immunostaining of a wide variety of biopsies was studied using different antigen retrieval fluids and heating and digesting systems (Pileri et al., 1997). This study showed the superiority of pressure cooking and EDTA over other methods, including microwave heating and proteolytic treatment.

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10. Ultrasound treatment was compared with microwave heating and pressure cooking;

11.

12.

13.

14.

15.

16.

17. 18.

the former treatment was claimed to be quantitatively and statistically superior for the immunostaining of prostatic basal cell structural cytokeratins using monoclonal antibody K8.12 (Portiansky and Gimeno, 1996). Compared with trypsinization–microwave heating, microwave heating– trypsinization demonstrated optimal immunostaining of Ki-67 using monoclonal antibody MIB-1 (Szekeres et al., 1995). Immunostaining using a panel of 21 antibodies was compared by employing microwave heating, microwave–pressure cooking, autoclave, and steamer (Taylor et al., 1996b). These methods yield similar intensities of staining provided the durations of heating are appropriately adjusted. Immunostaining of cytokeratin 18 in normal and neoplastic hepatocytes using antibody CK 18 was compared by employing microwave heating (15 min), autoclaving (10 min), pressurized boiling (1min), and simple boiling (15 min) in 10 mM citrate buffer (pH 6.0) (Xiao et al., 1996). No difference was found in the degree of immunostaining with light and electron microscopy. Compared to microwave heating, digestion with proteinase K for 2–4 min at room temperature yielded better retrieval of cytokeratins in mouse tissues using monoclonal antibodies (e.g., AE1, AE3) generated against human cytokeratins (Martin et al., 2001). Immunostaining of a number of proteins between microwave heating at 100°C of 20 min and boiling on a conventional hot plate. No difference was observed in the results of the two methods (Varma et al., 1999). Antigens bcl-2, CD3, and CD79a in tonsil tissue embedded in methyl methacrylate show superior immunostaining with trypsin followed by superheating at 121°C in a pressure cooker compared with that obtained with microwave heating only (Hand and Church 1998). Among the three antigen retrieval methods, hydrated autoclaving, microwave heating, and simple heating, simple heating overnight at 60°C was most effective for smooth muscle actin labeling (Igarashi et al., 1994). More intense and widely distributed staining of cytokeratins was observed with protease digestion than with microwave heating in benign lesions in the prostate using mouse monoclonal antibody (Googe et al., 1997).

Chapter 7

Antigen Retrieval on Resin Sections

Most commonly, antigen retrieval involves heating sections of paraffin-embedded tissues prior to light microscopy. However, antigen retrieval can also be accomplished on sections of resin-embedded tissues (Fig. 7.1). Tissues embedded in a resin show superior preservation of cellular details compared with those embedded in paraffin. Moreover, resin sections permit high resolutions to be obtained. In addition, semithin or thin (8–100 nm) sections can be obtained from resin-embedded tissues, allowing correlative studies using light and electron microscope, respectively (see Fig. 1.3). For details of resin microscopy and on-section immunocytochemistry, the reader is referred to Newman and Hobot (2001). The immunostaining quality of resin sections is usually comparable to that yielded by paraffin sections. Prior microwave heating of resin sections results in enhanced immunoreactivity with specific, easily interpretable staining using a variety of antibodies. In addition, resin sections allow immunogold and immunogold-silver immunostaining. Excess background staining is not a problem with resin sections, provided they are premicrowaved or heated by other means. In some cases, resin sections may show less intense staining than that exhibited by paraffin sections, which is due to thinness (~80 nm) of the former sections. Also, positive staining may not be achieved in some cases. This is exemplified by antibodies to neutrophil elastase and CD61, which show negative immunostaining on resin sections even after microwave heating (McCluggage et al., 1995). In contrast, immunostaining of CD20 is more reliable on resin sections than on paraffin sections of bone marrow trephine biopsy specimens. Note that the reaction of antibody with antigen is a surface phenomenon in resin sections. Various types of resins can be used for tissue embedding for antigen retrieval. Both water-miscible and water-immiscible resins (Hayat, 2000a) can be used in immunostaining for light and electron microscopy. Water-miscible resins used in light microscopy include the acrylic polymer glycol methacrylate (Suurmeijer and Boon, 1993b) and LR White (Sormunen and Leong, 1998), as well as the hydrophobic resins methyl methacrylate (Hand et al., 1996) and Polybed 812 (McCluggage et al., 1998). All these were used with prior microwave heating. Recently, using EDTA and heat, antigen retrieval was accomplished on Epon sections (Röcken and Roessner, 1999). The following embedding mixture is excellent when sections of thickness are required. It has been employed for embedding bone marrow trephine biopsy specimens for
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light microscopy (McCluggage et al., 1995):
Polarbed 812 DDSA MNA DMP-30 50 g 32 g 21 g 2g

DDSA: dodecenylsuccinic anhydride MNA: methyl nadic anhydride DMP-30: 2, 4, 6, tris (dimethylaminomethyl) phenol

ROLE OF FIXATIVE AND EMBEDDING RESIN IN ANTIGEN RETRIEVAL
It is well established that formaldehyde reacts with amino groups on protein side chains (Fig. 7.2), introducing mostly reversible protein crosslinks. Epoxy monomer reacts with the

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new hydroxyl groups introduced on the protein by formaldehyde; the epoxy molecules thereby are copolymerized with the protein. In other words, formaldehyde functions as a link between protein side groups and the epoxy monomer (Brorson et al., 1999). In contrast, such a copolymerization does not occur between acrylic resins and tissue proteins. These resins permeate the tissue without chemically binding to them. Accordingly, during thin sectioning, the two resins cleave differently. In the case of acrylic resins, the surface of cleavage tends to follow the path of least resistance; this path is the interface between the resin and proteins. Thus, more epitopes without splitting are exposed at the surface of acrylic sections. On the other hand, in the case of epoxy sections, the resistance in such interfaces is not significantly less than that in tissue proteins, which results in the splitting of protein

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molecules. Also, the surface of epoxy sections is smoother than that of acrylic sections. Consequently, fewer epitopes are exposed on the surface of the former sections. However, epoxy resins are of value, being easier to cut and more stable under the electron beam and better preserving the ultrastructure.

IMMUNOSTAINING OF THIN RESIN SECTIONS
Antigen sites can be unmasked not only on thick and semithin resin sections for light microscopy but also on thin resin sections for electron microscopy. Antigen retrieval at the ultrastructural level has been accomplished on thin sections of epoxy resins (Stirling and Graff, 1995; Röcken and Roessner, 1999) and LR White resin (Wilson et al., 1996; Sormunen and Leong, 1998). Because epoxy and LR White resins are superior to some other resins with respect to preserving the cellular details and other characteristics, antigen retrieval methods using these two resins for electron microscopy are presented. For electron microscopy, tissues can be fixed with a mixture of formaldehyde and glutaraldehyde or with the latter only. Glutaraldehyde fixation better preserves cellular details but strongly masks antigens. However, antigenic sites can be unmasked on epoxy thin sections of glutaraldehyde-fixed tissues by exposing the sections to strong oxidizing agents such as EDTA, hydrogen peroxide, sodium methoxide, or sodium metaperiodate. These treatments also allow immunostaining of sections of postosmicated tissues by removing osmium bonds. Moreover, such treatments temporarily minimize the hydrophobicity of epoxy section surface and may increase resistance to heavy metal poststaining (Bendayan and Zollinger, 1983; Causton, 1985; Newman and Hobot, 1993). The above-mentioned etching pretreatments are generally useful for epoxy sections but not for acrylic (LR White) sections because unlike acrylic resins, epoxy resins form covalent bonds with proteins. In other words, epoxy resins copolymerize with the tissue, while acrylic resins surround the tissue components without becoming part of them. Accordingly, epoxy resins strongly mask the proteins that become mostly inaccessible to antibodies. Therefore, epoxy sections, especially of glutaraldehyde-fixed tissues, require etching to unmask the antigens. The surface of acrylic sections is rougher than that of epoxy sections. Moreover, acrylic sections are less crosslinked and more hydrophilic than epoxy sections. As a result, immunostaining reagents penetrate acrylic sections easily, facilitating antigen detection. Exposure of acrylic sections to oxidizing agents worsen both the known instability of these sections under the electron beam and the structural details. To facilitate the access of antigens to antibodies, the protocol of embedding and etching given on page 159 is used (Crowley, 1997). The sections of this low-crosslinked embedding medium are thought to allow easy penetration of aqueous immunostaining fluids. A saturated solution of sodium periodate is prepared by dissolving 1 g of this reagent in 5 ml of distilled water and passing the solution through a pore filter. The grids are wetted by floating them on drops of distilled water and then floated on drops of the sodium periodate solution for ~15 min (this duration can be changed to obtain maximum immunoreactivity). The grids are thoroughly rinsed in distilled water and must not be allowed to dry before immunostaining.

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Embedding Media: Araldite 502 Eponate 12 DDSA Dibutyl Phthalate DMP-30

15ml 25ml 55ml 1% 1.5%

If background staining is a problem and standard rinsing with PBS fails to reduce nonspecific staining, boosting the sodium chloride concentration from ~0.9% (150 mM) to ~4.5% (750 mM) may help (Chiovetti, 1998). After this treatment, the grids must be rinsed several times in standard PBS before further processing, so that the salt concentration is reduced to the range of physiological strength. As an example, the grid can be rinsed five times for ~2 min each on drops of high-salt buffer, followed by two rinses for ~2 min each on drops of standard salt buffer. This routine can be used after incubation in the primary antibody or any other incubation (e.g., secondary antibody incubation, colloidal gold) that is suspected of contributing to nonspecific background staining. Although the exact explanation for the beneficial effect of the high-salt concentration is not known, it may alter the conformation of protein molecules and change their overall charge, making them less likely to bind nonspecifically on the surface of the section. It is known that high salt concentrations tend to precipitate proteins out of the solution in biochemical studies and are also used to wash chromatography columns. Accordingly, only the antibody molecules that have been bound specifically to antigenic sites remain on the section surface in the presence of high salt concentrations. If cross reactivity is a problem during conjugated gold-antibody double labeling with monoclonal antibodies from the same animal (e.g., mouse monoclonals), it can be avoided by incubating very carefully first one side of the grid in one of the mouse monoclonals and then the other side of the grid in the second mouse monoclonal (Chiovetti, 1998). Precaution must be used to prevent sinking of the grid in drops of the incubation reagents. Hexagonal mesh, uncoated nickel grids should be used. To avoid the adverse effect of high temperatures on thin resin sections in the microwave oven, staining can be carried out at ~5°C in the microwave oven (HernándezChavarría and Vargas-Montero, 2001). Heat generated by microwave irradiation is dissipated by this approach. Rapid staining is accomplished by molecular vibrations in the microwave oven, which induce molecular collisions leading to accelerated chemical reactions. Thin resin sections of the tissue fixed with glutaraldehyde/osmium tetroxide are transferred onto a grid, which is then placed into a BEEM capsule. Six capsules are placed on a plastic support, which is placed into a 500-ml beaker containing ice cubes and 300 ml of tap water, covering the bottom of the capsules. It takes ~5 min to equilibrate the temperature in the ice bath to 5°C, which is maintained during staining in the microwave oven. The temperature is measured after each heating period, and ice cubes are added as melting occurs. The staining is carried out with of 4% uranyl acetate in 50% ethanol for 1 min in a microwave oven set at a power level of 125.6W, followed by rinsing with 500 ml of distilled water. This is followed by staining for 1 min with of triple lead citrate (Sato et al., 1988) and then rinsing with 500 ml of distilled water. This lead citrate staining solution avoids the production of artifactual lead carbonate precipitates.

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ANTIGEN RETRIEVAL ON SECTIONS OF MODIFIED EPOXY RESIN
Because epoxy resins copolymerize with tissue proteins, and acrylic resins do not, sections of the former yield less immunostaining. However, to take advantage of the other superior characteristics of epoxy resins explained earlier, the immunostaining of sections of these resins can be enhanced by moderately increasing the proportion of the accelerator DMP-30 and microwave heating (Brorson, 1998a, b; Brorson et al., 1999). Conventional concentrations of accelerator in the epoxy mixture form abundant chemical bonds between resin and tissue. In contrast, a high concentration of accelerator reduces copolymerization of the epoxy resin with tissue proteins, while heating breaks down both protein crosslinkages introduced by aldehydes and the bonds between the resin and the tissue. The breakdown of abundant bonding with heating in the former case is insufficient to allow efficient access of the antibody to the antigen. Figure 7.3 shows the possible mechanism responsible for exposing epitopes to antibodies on the surface of thin epoxy sections after heating. Tissue specimens are fixed overnight at 4°C with a mixture of 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3). They are dehydrated in ethanol followed by propylene oxide. Infiltration is carried out in two steps using DMP-30 in concentrations of 4% and 2%, respectively, and embedding in the resin containing 2% DMP-30. The specimens in gelatin capsules are polymerized for 3 days at 56°C. Thin sections mounted on nickel grids are treated in 0.01 M citrate buffer (pH 6.0) for 15 min at 95°C in a PCR machine (GeneAmp 2400, Perkin Elmer). The sections are treated with 10% BSA in PBS (pH 7.2) for 4 hr to block nonspecific labeling. Incubation is carried out overnight at 4°C in the primary antibody, appropriately diluted in PBS. This is followed by washing three times for 5 min each in PBS and incubation for at 22°C in colloidal gold (15 nm)–conjugated secondary antibody, appropriately diluted in PBS containing 3% BSA. The sections are poststained with 5% uranyl

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acetate in 30% ethanol for 20 min and then with lead citrate for l0 min. The results of this procedure are shown in Figure 7.4.

EFFECT OF HEATING
Heating is effective in antigen retrieval on semithin and thin sections of resin-embedded tissues. This results not only from the breakdown of protein crosslinks introduced by aldehyde but also from the breakage of bonds between the epoxy resin and the embedded tissue (see Fig. 7.2). It is known that epoxy resins form covalent bonds with tissue proteins during embedding. Microwave heating has been employed for antigen retrieval on thin sections of formaldehyde and tissues embedded in Araldite for electron microscopy (Stirling and Graff, 1995). In this study thin sections on grids were treated for 1 hr at room temperature in a humid chamber with a saturated aqueous solution of sodium metaperiodate to reverse the effects of The heat treatment was carried out on a hot plate. Treatment of thin sections with sodium ethoxide is not recommended, for it damages the ultrastructure. Microwave heating has also been used for antigen retrieval on thin sections of tissues fixed with glutaraldehyde and and embedded in LR White or TAAB resin (Wilson et al., 1996). In this study, compared with nonmicrowaved sections, microwave-treated thin sections revealed markedly enhanced gold labeling of type IV collagen in the oral epithelial basal lamina for both types of resins.

ANTIGEN RETRIEVAL ON THIN RESIN SECTIONS USING AUTOCLAVING
In addition to antigen retrieval on sections of paraffin-embedded tissues for light microscopy, antigen retrieval can be carried out on thin resin sections for electron

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microscopy. Using heat pretreatment, antigen retrieval can be accomplished on paraffin sections and thin resin sections. The following method was used for immunostaining thin sections of tissue embedded in a resin (Xiao et al., 1996). Tissues are fixed with a mixture of 3% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 6 hr at 20°C. They are embedded in Lowicryl K4M at –40°C. Thin sections are mounted on uncoated metal grids or synthetic grids and airdried. They are placed in 10 mM sodium citrate buffer (pH 6.0) and heated in an autoclave for l0 min at 120°C. After being cooked for 20–30 min at room temperature, the sections are rinsed in PBS (pH 7.4). The sections are immersed in PBS containing 0.1% BSA and 0.1% gelatin (PBSG) for 5 min, and then treated with 10% normal goat serum in PBSG for 10 min. This is followed by incubation in the primary antibody (appropriately diluted) in a humid chamber overnight at 4°C. The sections are rinsed in PBSG containing 0.1% Tween 20 and incubated for 1 hr in colloidal gold (15 nm)–labeled goat antimouse IgG diluted in 1:20 with PBSG containing

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0.1% Tween 20. Following rinsing in PBSG containing Tween 20, the sections are rinsed in PBSG and then in PBS. The sections are postfixed for l0 min with 2% glutaraldehyde in PBS and rinsed several times in PBS and distilled water; poststaining is carried out with uranyl acetate and lead citrate. For negative controls, the primary antibody is replaced with PBS. The results of this procedure are shown in Figure 7.5.

RAPID STAINING OF THIN RESIN SECTIONS IN MICROWAVE OVEN
A microwave oven operating at 2,450 MHz with a maximum output power of 900 W and a cycle time of 2 sec can be used for rapid staining in a microwave oven (Cavusoglu et al., 1998). The oven contains a gas exhaust system and a built-in ceramic thermocouple temperature probe (PT 100). To determine the distribution of microwave heating, a piece of thermal paper is placed on the floor of the oven and subjected to microwave heating at 900 W for 1 min, and the hot spots are located. A glass bottle containing 40 ml of tap water is placed in the oven to measure the temperature during heating. The temperature of the water is monitored throughout the staining. Tissues are fixed with glutaraldehyde followed by and embedded in Epon. Thin sections are mounted onto a Formvar-coated grid, which is placed (section side down) on the surface of 4% aqueous solution of uranyl acetate in a staining dish. The dish is placed on the hot spot in the oven at a power of 600 W for 1 min at 20°C (initial temperature) to 94°C (final temperature). The dish is taken out of the oven and the grid is rinsed with distilled water. The grid is placed on the surface of lead citrate solution in the dish, which is placed in the oven and stained for 1 min at 20°C (initial temperature) to 93°C (final temperature). The results of this procedure are shown in Figure 7.6 (Cavusoglu et al., 1998). Figure 7.7 shows ultrarapid staining of biopsy heart tissue with uranyl acetate and lead citrate for 15 sec each in a microwave oven.

MICROWAVE HEAT-ASSISTED RAPID PROCESSING OF TISSUES FOR ELECTRON MICROSCOPY
In certain diagnostic studies with the electron microscope, it is helpful to complete fixation and embedding as quickly as possible. This accelerated processing can be completed in ~2 hr. As shown in Figure 1.1B, the quality of cell preservation is satisfactory. The recommended protocols for routine processing, routine microwave processing, and vacuum microwave processing, respectively, are given in Table 7.1 (Giberson et al., 1997).

MICROWAVE HEAT–ASSISTED IMMUNOLABELING OF RESIN-EMBEDDED SECTIONS
Conventional, high-resolution immunoelectron microscopy has been extensively used for the subcellular distribution of proteins to obtain information on their functions. However, this approach is time consuming. Processing time can be substantially reduced by applying microwave heating; the total time is reduced to 4–5 hr while the conventional

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method requires ~20 hr. All steps are carried out in a microwave oven. Tissues are fixed in a mixture of formaldehyde and glutaraldehyde, dehydrated in ethanol, and embedded in LR White resin for 75 min. Thin sections are incubated in primary antibody at 37°C for 15 min and then in colloidal gold–goat antirabbit IgG for 15 min at the same temperature (Rangell and Keller, 2000). Temperature should be strictly controlled in the microwave oven with a temperature probe that has a feedback mechanism to regulate the energy output of the microwave oven and thus maintains the optimal temperature. Alternatively, temperature can be controlled by placing a water load in the chamber of the microwave oven, which absorbs extra energy and provides humidity, slowing the evaporation of reagents. In addition, hot spots in the chamber should be avoided by using the neon bulb display method (Chapter 5). Labeling in the microwave oven is usually carried out at 37°C for 15 min. Longer durations and higher temperatures may result in undesirable changes in antibody concentration and molarity of the salts and pH. After heat treatment, the sections should be kept at room temperature for at least 2 min to stabilize the antibody-antigen complexes. The step-by-step procedure for microwave heat-assisted immunolabeling of resin-embedded thin sections for electron microscopy follows (Rangell and Keller, 2000):
1. Fix the tissue in a mixture of 2% formaldehyde and 0.3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 40 sec at 37°C in a microwave oven. 2. Rinse twice for 2 min each in the buffer at room temperature. 3. Dehydrate twice for 45 sec each at 45°C in a microwave oven. 4. Infiltrate for 15 min at 50°C with 1:1 mixture of 100% ethanol and LR white resin in a microwave oven. 5. Infiltrate three times for 10 min each at 50°C in a microwave oven. 6. Polymerize for 15 min at 95°C in a microwave oven using the temperature probe. 7. Polymerize for 1 hr at 95°C in a microwave oven without using the temperature probe. 8. Cut thin sections (may require 30 min). 9. Transfer sections onto grids and float on drops of PBS containing 0.1% Tween-20 (PBST) for 5 min at 37°C in a microwave oven. 10. Float the grids on drops of PBST containing 1 % bovine serum albumin and 0.1% coldwater fish skin gelatin (PBST+BG) for 5 min at 37°C in a microwave oven. 11. Incubate by floating sections three times for 5 min each at 37°C on drops of the primary antibody in PBST+BG in a microwave oven. 12. Keep the sections for 2 min at 37°C in a microwave oven to stabilize antibodyantigen complexes. 13. Rinse twice for 5 min each in PBST+BG at room temperature. 14. Incubate three times for 5 min each at 37°C on drops of the secondary antibodycolloidal gold complex in PBST+BG in a microwave oven. 15. Keep the sections for 2 min at 37°C in a microwave oven. 16. Rinse twice for 5 min each in PBST at room temperature. 17. Rinse twice for 5 min each in double-distilled water at room temperature. 18. Stain in 1% uranyl acetate for 30 sec at 37°C in a microwave oven. 19. Rinse twice for 5 min each in double-distilled water at room temperature. The total duration is ~4.3 hr.

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MICROWAVE HEAT-ASSISTED IMMUNOGOLD METHODS
Both immunogold alone and silver-enhanced immunogold methods can be employed in combination with microwave heating for labeling antigens. The use of colloidal gold particles as markers for antigenicity has become truly universal (Hayat, 1989–1991, 2000a). The advantage of the immunogold–silver staining (IGSS) method over the immunogold technique is that the former allows the use of very small colloidal gold particles for detecting antigenicity with both light and electron microscopy (transmission and scanning electron microscopy) (Hayat, 1995). These methods are significantly more sensitive than standard immunoperoxidase procedures. The major benefit of combining immunogold methods with microwave heating is shortening the duration of incubation in the primary and secondary antibodies. The use of microwave heating also offers the potential for increasing the positive reaction product and decreasing the nonspecific background label. The immunogold method can also be employed in conjunction with EDTA and conventional heat for electron microscopy (Röcken and Roessner, 1999). In this study human autopsy tissue specimens were fixed with a mixture of 2% formaldehyde and 2.5% glutaraldehyde and embedded in Epon. Various etching and antigen retrieval techniques were tested. The ideal pretreatment for achieving increased immunogold staining of amyloid consisted of conventional heating of thin resin sections at 91 °C for 30 min in 1 mM EDTA (pH 8.0).

Immunogold-Silver Staining
Jackson et al. (1988) were the first to employ the immunogold–silver staining (IGSS) method in combination with microwave heating. They completed within minutes the incubations in primary and secondary antibodies for detecting immunoglobulins in paraffin sections of human tonsil. van de Kant et al. (1990) applied the same method, except that resin instead of paraffin sections were used to detect bromodeoxyuridine incorporated in cells of the mouse testis. Tissue morphology is preserved better in a resin than in paraffin. The former also allows the use of thinner sections. Boon et al. (1989) used a similar procedure for staining beta-human chorionic gonadotropin in paraffin sections of the syncytiotrophoblast of first-trimester placenta. Recently, using the IGSS method, Taban and Cathieni (1995) visualized the goldprotein-ligand complex on cryostat sections of rat brain; this method can be used for light and electron microscopy.

Droplet Procedure
Tissues are fixed with buffered formalin or Kryofix and embedded in paraffin (Boon et al., 1991). Sections ( thick) are transferred to a glass slide, deparaffinized, rehydrated, and washed in running tap water for l0 min. They are treated with Lugol’s iodine for 5 min and rinsed briefly in tap water. Following destaining with 2% aqueous sodium thiosulfate for 10–15 sec, the sections are washed in running tap water for l0 min.

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The sections are washed in two changes of 5 min each in Tris buffer I (2.5% NaCl and 0.55% Tween 20, diluted in 0.05 mol/liter Tris-HCl buffer, pH 8.2). The excess buffer is removed by wiping around the sections, which are then covered for l0 min with of normal goat serum (NGS) diluted 1:1 with PBS (pH 7.4). Excess NGF is removed by wiping around the sections. The sections are covered with of primary antibody appropriately diluted in PBS (pH 7.4) containing 0.05% BSA (freshly prepared). The slide is placed on the polystyrene platform in the microwave oven and heated at 50% power for 5 min. A water load of 200 ml tap water has already been placed in the oven. The sections are washed with Tris buffer for l0 min, followed by washing in two changes of l0 min each in Tris buffer II (0.05 mol/liter Tris-HCl buffer, pH 8.2). Excess buffer is removed by wiping around the sections, which are then covered with of NGS for 10 min at room temperature. Excess NGS is removed, and the sections are covered with of colloidal gold conjugated secondary antibody. The slide is placed on the polystyrene platform in the microwave oven and heated at 50% for 5 min; the oven contains a water load of 200 ml of tap water. The sections are washed in three changes of 5 min each in Tris buffer II, rinsed with distilled water, and washed three times of 3 min each in distilled water. After excess water is removed, the sections are covered with of silver enhancement mixture for 8–11 min at 20°C. The silver enhancement mixture is prepared immediately before use by mixing equal volumes of the enhancer and initiator solutions of the Janssen Intense™ LM kit. The sections are washed three times for 5 min in distilled water, dehydrated, and mounted.

Chapter 8

General Methods of Antigen Retrieval

GENERAL PROCEDURE FOR ANTIGEN RETRIEVAL USING MICROWAVE HEATING
(See Fig. 8.1.) 1. Fix the tissue without delay for 4–6 hr in 4% buffered formalin; the longer the tissue remains in the fixative, the lesser the chances of epitope retrieval (Hayat, 2000a,b). 2. Wash in several changes of PBS; if available, an Autotecnicon should be used in this step and in steps 3 and 4 below. 3. Dehydrate in a series of ascending concentrations of ethanol. 4. Infiltrate and embed in paraffin. sections with a microtome and float them on a water bath kept at 5. Cut room temperature so as to stretch the sections. An ordinary glass slide is used to transfer the sections onto another water bath kept at 58°C to further stretch the sections. Lift them by the SuperFrost slides, thus mounting them in the process. The sections are allowed to dry in an upright position in a slide holder at a temperature of <30°C. When the slide holder is full, it is transferred to a conventional oven. 6. Dry the sections overnight at 58°C in the oven. 7. Remove the sections from the oven and deparaffinize them with three changes of 5 min each: in xylene, followed by three changes of 100% ethanol, two changes of 75% ethanol, and three changes of distilled water. A wash in Tris-buffered saline (TBS) (pH 7.3) for l0 min is optional. 8. Place the slides in a microwave-proof (microwave-transparent) jar containing 0.01 M sodium citrate buffer (pH 6.0); rectangular plastic jars are better than glass Coplin jars. Plastic Coplin jars are commercially available (Baxter Scientific, S7666). When vacuum is used in the microwave oven, use Teflon jars with thick walls or high-quality glass jars instead of plastic jars. This jar is kept in another larger jar containing water to catch the boil-over from the smaller jar containing the slides. Place a jar containing the buffer or distilled water in the oven during the boiling of the slides so that when required to top up the slide jar,
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the liquid is at the same temperature as that of the slides. This jar also acts as neutral ballast in the oven, slowing down the speed at which the boiling temperature is achieved. The buffer is prepared as follows: Stock solution A: 0.1 M citric acid is prepared by mixing 21.01 g citric acid with enough distilled water to make 1,000 ml. Stock solution B: 0.1M sodium citrate is prepared by mixing 29.41 g sodium citrate with distilled water to make 1,000 ml. The working solution is prepared just before use by mixing 18 ml of solution A with 82 ml of solution B plus sufficient distilled water to make 1,000 ml, and adjusting the pH to 6.0. The jar containing the slides can be covered with loosely fitting lids or vented screw caps; do not tightly close the jar or use aluminum foil to cover the jar. However, covering the jar is not obligatory if it has enough (2–3 cm) empty space above the buffer level. 9. Place the jars in the center of the oven on a rotary plate to ensure uniform heating of the slides. 10. Set the power to maximum; a power setting from 7–10 is recommended. 11. Set the time to 10–15 min, and check the buffer temperature with a temperature probe. The temperature of the buffer is different from that in the oven, so it is difficult to measure and control the temperature in the jar. 12. When the buffer begins to boil, allow it to cool for 5 min. Count the time of antigen unmasking from the boiling time. It is necessary to obtain vigorous boiling. The time for epitope unmasking depends upon the antigen, the antibody, the tissue type, the type of the fixative, and the duration of fixation. Thus, one has to standardize microwave heating by trial and error. A known positive control is essential. As an average, the time for epitope unmasking varies from 2–10 min. 13. Check the level of the buffer in the jar, and restore it between heating cycles. Compensate the buffer outflow with the buffer, while replenishing evaporated buffer with distilled water. The slides must remain fully immersed in the buffer. 14. After 5 min, again set the time to 5 min and restart the oven. 15. Repeat steps 12 and 13. 16. Remove the jar from the oven, and allow it to cool at room temperature for 20 min in a fume hood. 17. Rinse several times in 0.05 M PBS (pH 7.5). 18. Discard the used buffer. 19. When the DAB method is used, inhibit endogenous peroxidase by treating the sections with of 30% in 50 ml of PBS for 30 min, followed by thorough washing in PBS. 20. Treat the sections with a mixture of 3% normal serum and 0.4% Triton X-100 for ~30–60 min at room temperature to aid antibody penetration and block background staining. 21. Drain the excess serum from the slides, and incubate overnight at 4°C in a humid chamber with the primary antibody diluted appropriately in PBS. Incubation should be carried out with stirring to promote antigen-antibody contact. 22. Rinse in three changes of PBS.

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23. Incubate for 30 min at room temperature in the linking agent (biotinylated antiimmunoglobulin; Vector Laboratories, Burlingame, CA). 24. Rinse in three changes of PBS. 25. Incubate for 45–60 min in avidin-biotin peroxidase or alkaline phosphatase or ABC Elite or a third antibody if using the double indirect method. 26. Rinse in three changes of PBS. 27. Develop the color in 0.02–0.05% DAB activated with 0.003–0.01% in 0.1 M Tris buffer for 5–15 min. This step must be carried out in a fumehood. If using the alkaline phosphatase method, use the Alkaline Phosphatase Developing kit (Vector Red). 28. Wash in running tap water for 10 min. 29. Counterstain lightly for 20 sec with hematoxylin, and rinse in water. 30. Dehydrate in ethanol, clear in xylene, and cover-slip with Permount.

Note: Instead of coating the slides with an adhesive, use SuperFrost Plus slides (Fisher Scientific). These slides are charged positively and the sections are charged negatively, thus, preventing the sections from detaching from the slides while boiling. If these slides are unavailable, ordinary glass slides can be coated with an adhesive such as poly-L-lysine (0.1%) or neoprene (Aldrich Chemical, Milwaukee, WI). However, the use of any type of adhesive on the slide may not prevent detachment of sections from the slide. The main reason for losing sections during boiling is the presence of air bubbles between the section and the slide, not the lack of adherence of the section to the slide. To avoid the risk of drying the sections during microwave heating, it is necessary to heat them in multiple 4- to 5-min cycles and to replenish the jars between heating periods. Some evidence indicates that drying of sections on glass slides prior to histological staining in a microwave oven instead of in a conventional oven or on a hot plate, has several advantages: paraffin sections adhere better to the glue-coated slides, drying time is reduced from 1 hr to 1 min, and nonspecific background staining may be reduced. It has been suggested that drying of paraffin sections first at 38°C and then at higher temperatures improves immunostaining of the proliferating cell nuclear antigen (Golick and Rice, 1992). Additional studies are required to evaluate the relationship between the temperature of slide drying and the extent of immunostaining. Another suggestion is that mild boiling of the epitope retrieval fluid gently affects tissue sections, so they are less likely to be dislodged from the slide. The microwave oven should be left at high rather than changed to a medium setting because a change of setting does not affect the wavelength or actual power of the microwaves generated. It is thought that the intensity of specific immunostaining can be enhanced and background staining simultaneously reduced by gentle orbital rotation (using a serological rotation) of slides during manual incubations (Butz et al., 1994). Another advantage of this approach is shortening antibody incubation times without sacrificing sensitivity. Extreme antigen enhancement may cause false-positive staining. Such staining has been observed in the case of p53 antigen (using monoclonal antibody D07) with Target Unmasking Fluid (TUF) containing 35% urea in the microwave oven at 96°C for 30 min (Baas et al., 1996). This and other evidence indicates that there is a limit to the extent to which antigen enhancement can be applied to achieve optimal detection of a given antigen. If necessary, the slides with sections of paraffin-embedded tissues can be reused to detect a second type of antigen when the staining of the first type of antigen is negative.

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This need may arise when a limited number of slides is available. For this type of study, heat-induced antigen retrieval should be carried out only once. One such treatment lasts for many months. Repeated use of antigen retrieval tends to cause background staining. Such an approach has been successfully used for staining epithelial membrane antigens and cytokeratin in the tonsil tissue fixed with Bouin’s fixative (Roche et al., 2000).

ANTIGEN RETRIEVAL IN ARCHIVAL TISSUES
Storage of tissues, especially nervous tissue, in the fixative impedes immunostaining. It is often difficult to obtain postmortem and other human tissues that have been in the fixative for a short duration. Although some antigens, such as neuropeptide Y, in brain tissue are resistant to long fixation times, the reactivity of many other antigens in this and other tissues is significantly decreased when the fixation duration is longer than a few days because of excessive protein crosslinking. Examples of antigens sensitive to the effects of a long fixation time are parvalbumin, calbindin D28-K, MAP-2, MAP-5, and the nonphosphorylated part of the neurofilament. Physical and chemical changes such as temporary or permanent masking of epitopes occur in tissues during routine histological processing, including formalin fixation, alcohol dehydration, rehydration, and paraffin embedding. These changes are compounded in archival, paraffin-embedded specimens, which cause considerable loss of immunoreactivity. However, the problem is not as bleak as it may seem. Temporary loss of antigenicity due to overfixation can be retrieved by heating. In fact, at least certain types of antigenicities can be retrieved with heating irrespective of the length of fixation. Some of the diagnostically and prognostically important epitopes can be detected even in specimens stored in formalin as long as 60 years (Cattoretti et al., 1992). Either microwave heating or autoclaving is effective in epitope retrieval. Autoclaving at 120°C for 15 min, for example, is effective for the immunostaining of steroid hormone receptors (androgen, estrogen, and progesterone) in paraffin-embedded tissue sections stored for 10 years (Ehara et al., 1996). Another example is the study of epitopes (biomarkers) in archival paraffin blocks containing diseased tissue such as tumors, which is important for both identifying and characterizing early preinvasive neoplastic lesions, for correlating their expressions with diagnosis and prognosis of invasive tumors, and for investigating normal cellular activity (Grizzle et al., 1995; Myers et al., 1995). Identification of epitopes in archival tissues is also helpful in identifying patients who are eligible for novel therapies, including gene therapy and immunotherapy, and monitoring the effectiveness of conventional and novel therapies (Deshane et al., 1994). To accomplish the aforementioned and other antigenicity detection goals, epitopes must be detected reliably and reproducibly in archival, paraffin-embedded tissues. Since fixation is the most important factor in preserving and masking antigenicity in archival and other specimens, a brief comment on the effects of various fixatives on epitopes is relevant. There is no universally ideal fixative to optimally detect all types of epitopes in archival tissues. A few examples suffice. Immunohistochemical studies demonstrate that unbuffered zinc formalin (a slow crosslinking fixative) and unbuffered acid formalin yield good preservation of antigens p185 and TGF, whereas ethanol and methanol (two coagulating fixatives) produce good

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results for keratin and p53 antigens in 3-mm-thick paraffin sections of archival tissues (Arnold et al., 1996). Acid formalin is less effective as a protein crosslinking agent, as it protonates amino groups and thus allows easy access of the antibodies to the epitopes. In this study, buffered formalin produced the least satisfactory results, which was expected because the fixative forms protein crosslinks more rapidly and effectively. Nevertheless, note that the above-mentioned fixatives, other than buffered formalin, produce comparatively poor morphological details.

Method for Microwave Heating of Archival Tissue Blocks
The following method is recommended for immunostaining of MAP-2, SMI-32, SMI-311, SMI-312, and the calcium-binding proteins calbindin D28-K, parvalbumin, and calretinin in the neuroscience field (Evers and Uylings, 1994a, b, 1997). Human brains 6hr after death are fixed with 4% buffered formaldehyde for 4 years. Cortical blocks (5 mm), cut from the brain, are washed for several hours in running tap water and left in Tris-buffered saline (PBS) (pH 9.0). They are transferred to a plastic jar containing PBS placed in a microwave oven, and heated at full power (boiling) for 15 min; the fluid level is checked every 5 min. The temperature is controlled with the temperature probe. It takes ~3 min to reach a temperature of 90°C. The jar is allowed to cool for 15 min, and then the tissue blocks are placed in TBS (pH 7.6). Thick sections are cut on a vibratome, collected in plastic vials containing TBS, washed several times in TBS for 1 hr, and immunostained according to standard procedures given in this volume. These thick sections allow staining of whole neurons, including neuronal processes, to distinguish different morphological types.

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ANTIGEN RETRIEVAL USING A CONVENTIONAL OVEN
Excessive treatment with microwave heating or enzymatic digestion tends to damage cell morphology and even occasionally destroy tissues, whereas insufficient treatment may result in false-negative results. Optimization of microwave heating is difficult. An alternative simple approach is to heat the deparaffinized sections overnight in l0mM citrate buffer (pH 6.0) in a standard oven at 70–80°C (Man and Tavassoli, 1996). A variety of nuclear and cytoplasmic epitopes (e.g., estrogen and androgen receptors, p 53, Ki-67, and cytokeratin) can be demonstrated with excellent perservation of morphology, intense reactivities, and clean background. Optimal immunostaining for each of the 15 antibodies tested was observed even when sections from 15 different cases were heated together for a fixed duration and then immunostained in an identical manner according to manufacturers’ recommended antibody concentrations. Thus, this method is a standard method for at least these 15 antibodies. Controls included omission of the primary or secondary antibody and substitution of the primary antibody with nonimmune sera or PBS. No positive immunostaining was observed in any of the negative controls. The only drawback of this approach is that it takes longer to yield results. The results of this method are shown in Figure 8.2 (Plate 3G).

HOT PLATE–ASSISTED ANTIGEN RETRIEVAL
In certain cases antigen retrieval in formalin-fixed and paraffin-embedded tissues is more efficient using conventional heating with a hot plate than that using standard microwave heating or pepsin predigestion. In such cases, the hot plate method yields stronger immunostaining even in tissues that have been fixed with formalin for as long as 1 month. This advantage is important when tissues have to be stored in formalin on a weekend or during holidays or transported to a service laboratory. The use of hot plate heating becomes essential when the diagnosis of malignancy is based on a negative immunoreaction. This is exemplified by the malignant prostate gland. It is known that anti-high-molecular-weight cytokeratin (HMCK) monoclonal antibody clone selectively stains the basal layer of the prostatic duct–acinar system in the benign prostate. In contrast, malignant glands lack immunoreactivity with this antibody (Wojno and Epstein, 1995). This antibody is especially sensitive to formalin fixation and different antigen retrieval methods. A recent study demonstrates that hot plate heating is better than other antigen retrieval methods for detecting even weak HMCK positive staining in the radical prostatectomy specimens fixed with formalin for 6 hr to 1 month (Varma et al., 1999). Although formalin fixation for 6 hr is the optimal duration of fixation, there is no decrease in HMCK immunoreactivity in tissue fixed for 1 month when hot plate heating is used. This advantage is shown in Figure 8.3; the cells were fixed for 54 hr. The above and other evidence reinforce the necessity for standardized fixation and antigen retrieval method in each laboratory.

Procedure
Sections thick) of formalin-fixed and paraffin-embedded tissues are mounted on gelatin-coated slides, which are placed in a beaker containing 1,000 ml of 0.2 M sodium

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citrate buffer (pH 6.0) and heated for l0 min at 100°C on a hot plate (PC-351) (Corning, Utica, NY). The slides are allowed to cool at room temperature for 20 min. After being washed in PBS, the sections are incubated overnight at 4°C in the monoclonal antibody clone E12 (DAKO, Carpinteria, CA) and diluted 1:4000 with PBS. They are washed with PBS and then treated with the Elite ABC system according to the directions of the manufacturer (Vector, Burlingame, CA). The chromogen is developed with 3-amino-9 ethyl carbazole (Sigma) at room temperature. After being washed with PBS, the sections are counterstained with Mayer’s hematoxylin and mounted in Glycergel (Sigma). The results of this procedure are shown in Figure 8.3.

HOT PLATE–ASSISTED GRADING OF VULVAR INTRAEPITHELIAL NEOPLASIA
The grade of vulvar intraepithelial neoplasia (VIN) can be scored by subclassifying it on the basis of the extent of cellular changes into three types (Van Beurden et al., 1999):
1. VIN 1 (cellular disarray involves the lower two-thirds of the epithelium) 2. VIN 2 (cellular disarray involves more than the lower two-thirds of the epithelium) 3. VIN 3 (cellular disarray involves more than the lower two-thirds of the array)

Vulvar intraepithelial neoplasia show a spectrum of pathological alterations, including nuclear pleomorphism, hyperchromasia, altered epithelial maturation, cellular aggregation, loss of normal keratinocyte polarity, and atypical mitotic features (Wilkinson, 1992).

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Conventional light microscopy of multifocal lesions of VIN may not show VIN 3 and instead may show VIN 2, VIN 1, or even normal squamous epithelium (van Beurden et al., 1998). Interobserver variation in the interpretation of the grading of VIN is not uncommon. To determine correct treatment of the patient, it is necessary to know which lesions show VIN 3 and which do not. The standard treatment for VIN 3 is drastic surgical excision of all visible lesions. However, an alternate approach is taking multiple biopsies; the removal of involved skin using cold knife surgery or laser vaporization without radical surgery (van Beurden et al., 1998). Interobserver variation in the grading of VIN has been observed when using hematoxylin-eosin only, MIB-1 monoclonal antibody alone, or combined hematoxylineosin and MIB-1 antibody (van Beurden et al., 1999). This antibody is the most versatile proliferation worker for the sections of formalin-fixed and paraffin-embedded tissues. Normal vulvar skin and VIN lesions are fixed with formalin and embedded in paraffin according to standard procedures. Following deparaffinization with xylene and rehydration with ethanol, the slides are placed in 0.01 M sodium citrate buffer (pH 6.0) and boiled for l0 min on a hot plate; after cooling for 20 min to room temperature the slides are subjected to three different treatments:
1. Staining with hematoxylin-eosin 2. Incubation in MIB-1 antibody 3. Incubation with a combination of MIB-1 antibody and hematoxylin-eosin

Uncertainty regarding the grading of VIN is significantly decreased when MIB-1 antibody is used.

WATER BATH HEAT–ASSISTED ANTIGEN RETRIEVAL
Although the microwave heat–assisted antigen retrieval method is widely used in clinical pathology, it has several limitations. The rise of temperature in the microwave oven is difficult to control. Unintentional, very high heating of sections in the oven is not uncommon. Thus, standardization of temperature in the oven becomes difficult. Very high temperatures tend to damage cell morphology. Microwave heating is especially damaging to free-floating sections and causes wrinkling, among other problems. Such sections can also be expelled from the antigen fluid during vigorous bubbling at high temperatures in the microwave oven. Limitations of microwave heating have also been discussed on page 142. One of the alternatives to the microwave heating method is the water bath heating technique. The latter is simple and inexpensive and allows precise temperature control (including uniform rise in temperature), which enables the achievement of reproducible antigen retrieval as well as other histological data. The water bath can be heated in a conventional oven; alternatively, heated water baths are commercially available. The subboiling water bath heating (80°C) method is effective in the antigen retrieval on free-floating sections, cryostat sections, and paraffin-embedded sections of tissues fixed with formaldehyde and/or glutaraldehyde (Jiao et al., 1999). The advantage of using glutaraldehyde is its excellent preservation of the ultrastructure using electron microscopy. The water bath heating technique can also be used for immunofluorescence microscopy. This technique is especially useful for processing free-floating sections of brain tissue. The following methods

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are recommended for immunostaining on thick and thin sections using light and electron microscopy, respectively, and also for cell smears.

Procedure for Electron Microscopy
The animal is fixed by vascular perfusion with a mixture of 3% paraformaldehyde and 0.1% glutaraldehyde in 0.15 M PBS (pH 7.4) according to standard procedures. The brain (or another organ of interest) is removed and tissue blocks containing the region of interest are refixed overnight with the same fixative. The blocks are agar-embedded and cut into sections with a vibratome. The sections are washed with PBS and then with saline containing 10mM sodium citrate (SSC) (pH 8.5). They are placed in SSC and heated in a water bath for 30 min at 76–80°C. The sections are cooled in SSC to room temperature for l0 min and then washed with PBS. The sections are treated with 1% sodium borohydride in PBS for 15 min and then thoroughly washed with PBS. They are incubated in 4% normal serum in PBS for 1 hr, using serum other than that used to generate the primary antiserum. For example, if primary antiserum from rabbit is to be used, tissue should be prerinsed in normal goat serum (NGS). In the following procedure, each step is followed by a rinse in PBS, unless otherwise indicated. The sections are incubated for ~72 hr at 4°C with primary antiserum in PBS containing 2% normal serum and 0.25% gum arabic (Sigma). The sections are then incubated overnight at 4°C with a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA), diluted 1:200. They are next incubated overnight at 4°C with avidin-biotinylated peroxidase complex (ABC) diluted 1:100 in PBS containing 0.25% gum arabic. Note that the peroxidase-antiperoxidase procedure can also be used. The bound peroxidase is visualized by reaction with a filtered solution of 0.05% DAB and 0.0005% hydrogen peroxide in PBS. After rinsing with 0.15 M sodium phosphate buffer (pH 7.4), the sections are postfixed with 0.25% osmium tetroxide in the same buffer for 1 hr and counterstained with 1% uranyl acetate in deionized water. The sections are dehydrated and flat-embedded in Epon according to standard procedures (Hayat, 2000a). Controls are processed as above, except that incubation in the absence of the primary antibody is carried out with PBS containing 2% normal serum and 0.015% Triton X-100. The sections are observed under a light microscope and representative DAB-labeled neurons, and neuronal processes in the region of interest are selected for study with the electron microscope. The sections are trimmed with a razor Wade into small pieces containing the immunoreactive neurons. Thin sections are cut for electron microscopy. The results of this procedure are shown in Figure 8.4.

Procedure for Light Microscopy
Human brain tissues, for example, are fixed postmortem with 10% formalin for 24–48 hr, dehydrated, and embedded in paraffin. Sections, cut with a rotary microtome, are mounted on coated glass slides. The sections are rinsed three times for 5 min each with 0.1 M sodium phosphate buffer (pH 7.4) and then transferred to 10–15 mM sodium citrate

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(pH 8.5–9.0; preheated to 80°C in a water bath). They are cooled to room temperature, followed by rinsing three times for 5 min each in the buffer. The sections are immersed in 0.3% nonfat dry milk in buffer containing 0.3% Triton X-100 and 0.01% sodium azide for 30–60 min. The sections are incubated in a primary antibody (diluted appropriately) for 72 hr at 4°C in a sealed humid chamber; the incubation is carried out by applying droplets of the antibody to the sections. After being rinsed in the buffer, the sections are incubated for 90 min in secondary antiserum diluted 1:50 with PBX (0.3% Triton X-100, 0.01% sodium azide and 0.1 M phosphate buffer) and then treated for 1 hr under agitation in peroxidaseantiperoxidase (PAP), diluted 1:100 with PBX, in a sealed humid chamber in both cases. The sections are rinsed sequentially with the buffer and then distilled water. They are incubated for 10 min with continuous agitation in 50ml of 0.05 M imidazole/0.05 M cacodylate buffer (pH 7.2) containing 50 mg of DAB, followed by an additional 10-min incubation after adding of 3% hydrogen peroxide with continuous agitation. After being washed in distilled water, the sections are placed in the buffer, dried, dehydrated, and cover-slipped with Permount. Note that the ABC procedure can also be used for immunolabeling. For assessing the antigen retrieval effectiveness, the staining intensities observed under the microscope can be divided into five grades: + + + +,+ + +,+ +,+ or – for

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very intense, intense, moderate, weak, or negative, respectively. These judgments are made qualitatively by comparing one section to another.

Procedure for Free-Floating Sections
Free-floating sections of paraformaldehyde-fixed tissues are rinsed three times for 5 min each in 0.1 M sodium phosphate buffer (pH 7.4) (Jiao et al., 1999). They are transferred to 10–15 mM sodium citrate buffer (pH 8.5–9.0) preheated in a water bath kept in a conventional oven at 80°C for 30 min. The sections are allowed to remain in this buffer for 30 min to cool to room temperature. Following rinsing three times for 5 min each in the same buffer, the sections are treated by immersion in 0.3–3% nonfat dry milk in 0.1 % sodium azide for 30–60 min. The sections are then incubated in the primary antibody, diluted with a mixture of 0.3% Triton X-100, 0.01% sodium azide, 0.1 M sodium phosphate buffer (pH 7.4) (PBX), and 5% normal horse serum for 72 hr at 4°C under constant agitation. The sections are rinsed in the same buffer and incubated for 90 min in secondary antiserum (Jackson ImmunoResearch Laboratories, West Grove, PA), diluted 1:50 with PBX5% normal horse serum. They are rinsed in 0.1 M sodium phosphate buffer and then incubated for 1 hr in peroxidase antiperoxidase (PAP), diluted 1:100 with PBX. The sections are rinsed three times for 5 min each in the same buffer followed by distilled water rinses. They are incubated for 10 min in 50 ml of 0.05 M imidazole/0.05 M cacodylate buffer (pH 7.2) containing 50 mg of DAB. This is followed by an additional 10 min incubation after adding All incubations are carried out under constant agitation. The sections are washed in distilled water, placed in the same buffer, mounted onto gelatin-coated slides, dried, dehydrated, and cover-slipped with Permount. Note that the ABC procedure can be used instead of the PAP procedure.

MICROWAVE HEAT–ASSISTED EVALUATION OF GLOBAL DNA HYPOMETHYLATION
Loss of methyl groups in DNA is not uncommon in human carcinomas such as colon adenomas and adenocarcinomas. A strong correlation is found between the malignant phenotype and DNA methylation. It is known that 5-methylcytidine (a spontaneous frequent site of C and T mutation) is involved in the control of gene expression in carcinogenesis and in tumor progression. Consequently, global DNA hypomethylation could induce protooncogene expression, whereas hypermethylation could silence tumor suppressor gene (Little and Wainwright, 1995). Monoclonal antibodies can be used to recognize the presence of a methyl group on the C of cytidine to investigate DNA methylation in situ. An immunohistochemical method has been reported for correlating the histopathological pattern with the immunostaining intensity of the nuclei (Hernández-Blazquez et al., 2000). Qualitative and quantitative differences can be observed and measured between the normal and malignant part of each tumor specimen. Morphologically altered nuclei display densely labeled spots within faintly labeled areas, whereas normal nuclei are darker and uniformly stained.

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Procedure
Malignant lesions and normal tissue biopsies (colon) are fixed with formalin and embedded in paraffin (Hernández-Blazquez et al., 2000). Paraffin sections thick) are deparaffinized, rehydrated, and rinsed in PBS. They are placed in 0.1 mM citrate buffer (pH 3.4), heated for 10 min in a microwave oven at full power (720 W), and washed in PBS. The slides are immersed in 2N HC1 for 2 hr at 37°C and then rinsed in PBS. The sections are covered with of hybridoma supernatant containing anti-5-MeCyd monoclonal antibody and incubated for 1 hr at room temperature with a biotinylated goat antimouse secondary antibody diluted 1:200 in PBS containing 0.1% BSA. The sections are rinsed in PBS and then treated for 30 min with streptavidin-peroxidase, diluted 1:100 with PBS-BSA. They are rinsed in PBS and treated with for 5 min. The PBS used for rinsing contains 0.1% Tween 20.

MICROWAVE HEAT–ASSISTED ENHANCED PEROXIDASE ONE-STEP METHOD
The enhanced peroxidase one-step (EPOS) method is considered superior to standard ABC technique in that the former is more sensitive than the latter. It is known that the Ki-67 antibody can only be used on fresh or frozen tissues, whereas the monoclonal antibody MIB-1, developed against a part of the Ki-67 antigen molecule, can be used on sections of formalin-fixed and paraffin-embedded tissues using antigen retrieval. Recently, EPOS Ki-67 antibodies were developed which consist of antibody molecules and horseradish peroxidase bound covalently to dextran (Bisgaad et al., 1993). This method has been applied for localizing PCNA and Ki-67 antigens (Tsutsumi et al., 1995). More recently, the EPOS protocol was compared with the standard ABC technique for detecting Ki-67 antigens in pituitary tumors (Turner et al., 1999). Both methods were applied in conjunction with microwave heating. This study demonstrates that the EPOS method is both more convenient and more accurate in showing the number of cells that have entered the cell cycle. It can be inferred that the EPOS approach, in addition, detects very small amounts of Ki-67 antigens present in the cells in early stage, which the ABC method does not detect. Thus, the EPOS antibody may identify those tumors that are potentially aggressive and require closer monitoring.

Procedure
Surgically removed tissues are fixed with 4% buffered formalin and embedded in paraffin (Turner et al., 1999). Sections thick) on slides are deparaffinized, rehydrated, and then treated with 3% to block endogenous peroxidase. They are placed in 0.1 M sodium citrate buffer (pH 6.0) and heated in a microwave oven. An EPOS rabbit antihuman Ki-67 antibody is applied as supplied (DAKO); it is ready to be used without any dilution. Color development is accomplished with metal-enhanced DAB for 15 min, followed by light counterstaining with hematoxylin. Quantification of Ki-67 antibody-labeled

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cells is performed with an image analysis system (VIDAS 21), and the tumor cell nuclear staining is recorded as the percentage of positive cells (labeling index).

MICROWAVE HEAT–ASSISTED IMMUNOSTAINING OF CELL SMEARS
Consistent and reliable immunostaining of cytological preparations can be obtained by ensuring that the following conditions are met (Leong et al., 1999b): (1) availability of sufficient specimens for examination, (2) appropriate panel of diagnostic antibodies, (3) preparation of thin and uniformly spread smears, (4) removal of proteinaceous background fluid and red blood cells, (5) use of optimal fixation, and (6) application of a sensitive method of immunostaining. The concentration of the reagents including antibodies and the duration of incubation for cytological smears are lower and shorter, respectively, than those used for paraffin sections. These conditions must be determined by trial and error for each type of new antibody employed. Thin, uniformly spread cell smears are prepared on glass slides and then rehydrated with normal saline for ~3 min. This is followed by air-drying for 24 hr and fixation with 0.1% formal saline (1,000ml of normal saline and 2.5ml of 40% formalin) for 2–14 hr; postfixation is accomplished with 95–100% ethanol for 10 min. The sections are heated in 10 mM citrate buffer (pH 6.0) in a microwave oven for ~5 min at boiling and then allowed to remain in the hot solution for another 5 min before being removed for immunostaining.

DOUBLE IMMUNOSTAINING USING MICROWAVE HEATING
The following method was used for double immunostaining of fast and slow skeletal muscle fibers in the same section (Carson et al., 1998). Muscle specimens (2–4 mm) are fixed in 10% neutral buffered formalin for 3 hr and then immersed in 0.1 M PBS containing 17% sucrose for 3 hr. They are embedded in paraffin, and 4 to sections are mounted onto Superfrost Plus slides. The sections are deparaffinized and placed in Target Unmasking Fluid (TUF) preheated in a microwave oven and maintained at 90°C for 15 min (without boiling) and slowly cooled to room temperature. The sections are rinsed three times for 3 min each in PBS and then placed in 3% hydrogen peroxide in PBS for 30 min to block endogenous peroxidase, followed by again rinsing three times in PBS. The sections are blocked with 10% nonimmune goat serum for 30 min at room temperature and then rinsed in PBS. They are incubated overnight at 4°C in monclonal antibody against MHC-I (diluted 1:20 in PBS) in a humid chamber, followed by rinsing three times in PBS. The sections are incubated for 1 hr in the goat antimouse biotinylated secondary antibody in a humid chamber. The sections are treated for 1 hr with streptavidin-alkaline phosphatase in a humid chamber and then washed in PBS. They are developed for 10 min in nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP). After washing in PBS, the sections are treated for 30 min in a double-staining enhancer (Zymed Laboratories, San Francisco, CA) to prevent the first stain from reacting with the second. They are thoroughly rinsed in distilled water followed by PBS. The sections are treated for 30 min with 10% nonimmune goat serum and then rinsed in PBS. This is followed by overnight incubation at 4°C in monoclonal antibody against

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MHC-II (diluted 1:10 in PBS) in a humid chamber and rinsing in PBS. The sections are incubated for 1 hr in the goat antimouse biotinylated secondary antibody in a humid chamber. They are treated for 1 hr with streptavidin–alkaline phosphatase in a humid chamber and then washed in PBS. The sections are treated with 3-amino-9-ethyl-carbazole (AEC) chromogen; the staining intensity is monitored microscopically. The staining duration is ~10 min. They are counterstained for 10 min in NBT/BCIP, washed in distilled water; and cover-slipped with an aqueous mounting medium. An immunostaining kit (HistostainSP) is commercially available (Zymed Laboratories, San Francisco, CA).

MICROWAVE HEAT–ENHANCED DOUBLE IMMUNOSTAINING OF NUCLEAR AND CYTOPLASMIC ANTIGENS
Simultaneous, double immunostaining of two antigens in single cells in sections of formalin-fixed and paraffin-embedded archival tissues can be carried out. This is accomplished by using microwave heating to detect otherwise undetectable nuclear antigens, followed by the labeled avidin-biotin (LSAB) procedure and the alkaline phosphatase (APAAP) protocol to detect cytoplasmic or membranous antigens (Bohle et al., 1997).

Procedure
Sections of formalin-fixed and paraffin-embedded tissues on glass slides are deparaffmized in xylene (10 min), acetone (10 min), acetone/TBS (1:1, 10 min), and TBS (pH 7.4, 10 min). The tissues are fixed with 4% formalin and can be stored for up to 10 years. Endogenous peroxidase is blocked in methanol containing 0.5% for 10 min. Microwave heating is carried out by placing the slides in microwave-proof tubes containing 0.1 M sodium citrate buffer (pH 6.0) and boiling for 5 min at the 800 W setting. The tubes are refilled, and the heating is repeated four times. The slides are allowed to cool to room temperature and washed in TBS. Incubation is carried out with primary antibody in a humid chamber for 30 min at room temperature. After being rinsed three times in TBS, the slides are incubated with rabbit antimouse antibody (1:200) in TBS for 30 min at the same temperature. They are rinsed three times in TBS and incubated with streptavidin/HRP P397 (DAKO) for 30 min at room temperature. Following rinsing three times in TBS, the slides are incubated for 4 min in DAB chromogen solution. Thereafter, of the second antibody is placed on the slide and incubated in a humid chamber for 30 min at room temperature. After being rinsed three times in TBS, the slides are incubated with rabbit antimouse (“link”) antibody (1:25) in RPMI 1640 Medium (Life Technologies, Faisley, Scotland) for 30 min at room temperature. They are rinsed three times in TBS and incubated with the APAAP-complex (DAKO, 1:25 in RPMI 1640 Medium) for 30 min at the same temperature. Following rinsing three times in TBS, the incubation with the APAAP-complex is repeated three times for 10 min each. After being rinsed 10 times in TBS, slide development is carried out in new fuchsin chromogen solution for 30 min. The results of this procedure are shown in Figure 8.5 (Plate 3H).

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MICROWAVE HEAT–ASSISTED IMMUNOHISTOCHEMICAL LOCALIZATION OF CYCLIN D1
Cyclin D1 (PRAD-1, bcl-1) protein plays an important role in the transition of cells from resting phase to DNA replication phase. This protein has oncogenic properties, and its overexpression is thought to play a role in tumorigenesis. Overexpression of cyclin D1 mRNA and protein has been demonstrated in a variety of lymphoid as well as nonlymphoid neoplasms (de Boer et al., 1995; Vasef et al., 1997a, b; Naitoh et al., 1995). The overexpression of cyclin D1 protein has also been demonstrated in neoplastic proliferating parathyroid tissue, adenomas, and nonneoplastic proliferating parathyroid gland, but seldom in normal parathyroid tissue (Vasef et al., 1999). It is suggested that PRAD-1 gene alteration is responsible for cyclin D1 protein overexpression in parathyroid hyperplasia. The mechanism underlying such gene alterations remains undefined. Although cyclin D1 is not useful in distinguishing parathyroid carcinomas from parathyroid adenomas, this protein is useful in distinguishing between hyperplasia and normal parathyroid glands in histologically ambiguous cases. The following immunohistochemical method is recommended for the localization of cyclin Dl in neoplastic parathyroid tissue (Vasef et al., 1999). Biopsy tissue specimens are fixed with formalin and embedded in paraffin. Sections thick) are mounted on silanated slides, heated at 56°C for 1 hr, deparaffinized in xylene, rehydrated in graded ethanols, and rinsed in distilled water. They are placed in 0.01 M citrate buffer (pH 6.0) and heated in a microwave oven for six cycles of 5 min each, followed by cooling at room temperature for 20 min. Endogenous peroxidase activity is blocked with hydrogen peroxide in distilled water for 8 min, and nonspecific background staining is prevented by treatment with nonimmune horse serum for 20 min.

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The sections are incubated overnight at 4°C in anticyclin D1 monclonal antibody (P2D11F11) (Novocastra/Vector, Burlingame, CA), diluted 1:10; this antibody does not show cross-reactivity with cyclin D2 protein. After rinsing with PBS, the reactivity is detected using mouse immunoglobulin G as the secondary antibody with an avidin-biotin procedure and DAB as the chromogen. Sections of normal tonsil and known cases of cyclin D1–positive mantle cell lymphoma are used as negative and positive external controls, respectively. Cases are interpreted as cyclin D1 positive if more than 10% of cells show positive nuclear staining, while cases with patchy clusters of positive cells are defined as focally positive. The results of this procedure are shown in Figure 8.6 (Plate 4A).

MICROWAVE HEAT–ASSISTED IMMUNOFLUORESCENCE STAINING OF TISSUE SECTIONS
Frozen sections, as well as sections of formaldehyde-fixed and paraffin-embedded tissues, can be used for direct or indirect immunofluorescence staining after antigen retrieval using microwave heating. Although frozen sections yield higher sensitivity than that obtained with paraffin sections, the latter approach is necessary for retrospective studies of archival specimens. Moreover, fresh tissue is not always available. Antigen retrieval using trypsin digestion in conjunction with indirect immunofluorescent staining in various tissues was first reported by Huang et al. (1976). The 2-hr digestion in this study was too severe a treatment and adversely affected cell morphology. An improved antigen retrieval procedure consists of microwave heating in urea (6 M), followed by indirect immunofluorescence staining (D’Ambra-Cabry et al., 1995). This method produces more intense immunofluorescence staining than does trypsinization. Antigen retrieval can also be obtained by combining trypsin digestion with microwave heating, followed by direct immunofluorescent staining (Al-Rifai et al., 1997). This procedure is given below.

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Procedure
Sections thick) of formalin-fixed and paraffin-embedded tissues are deposited on a coated slide, deparaffinized, rehydrated, and rinsed in PBS. They are placed in sodium citrate buffer-containing plastic jars and heated twice for 5 min each in a microwave oven. Following cooling at room temperature for 20 min, the sections are treated with 0.3% trypsin. They are washed in PBS, blocked with normal serum for 10 min, and then overlaid with fluorescein-conjugated rabbit antibodies to human IgG at 1:20 dilution in PBS for ~45 min at room temperature. The sections are washed in PBS for 5 min and incubated for ~30 min in fluorescein isothiocyanate (FICT)-labeled swine antirabbit immunoglobulin conjugate at 1:20 dilution in PBS. After being washed in PBS for 5 min, the sections are mounted with an aqueous mounting medium. The sections are observed under an epiilluminating fluorescent microscope. The negative control is not incubated with the primary antibody, and a frozen section from a patient with the known condition is used as the positive control.

MICROWAVE HEAT–ASSISTED DOUBLE IMMUNOFLUORESCENCE LABELING
Most applications of immunohistological labeling to routine tissue sections focus on a single antigen. However, some studies require simultaneous labeling of two antigens. Double labeling, for example, is required to determine if two antigenic markers are expressed in the same cell and/or two antigens are present at the same site such as two markers at the cell surface. The enzyme-based methods (peroxidase and alkaline phosphatase) are rarely suitable for detecting two antigens present at the same site because one label tends to obscure the other. The reaction product of the antigen of a higher density may mask the reaction product of the antigen of a lower density. Consequently, these methods are largely restricted to the detection of pairs of antigens found either at different sites within a single cell (e.g., nucleus and cell surface) or in different cell populations. Double immunofluorescence labeling in conjunction with microwave heating can be used to visualize two markers at the same cellular location in routine formalin-fixed and paraffin-embedded tissue sections (Mason et al., 2000). The primary antibodies are either monoclonal antibodies of differing isotype/subclass or antibodies from different species. Labeling is visualized on a conventional fluorescence microscope equipped with a cooled analog monochrome CCD camera (Model C 5985, Hamamatsu Photonics, Billerica, MA) and recorded using “off the shelf” personal computer hardware and software. Contrary to general belief, paraffin-embedded tissue sections do not show excessive nonspecific fluorescence.

Procedure
Tissues are fixed in formalin, embedded in paraffin, and sections thick) are transferred onto Superfrost Plus–coated slides (Mason et al., 2000). The sections are deparaffinized with xylene and then rehydrated with descending concentrations of ethanol. They are placed in 0.1 M sodium citrate buffer (pH 6.0) and heated in a microwave oven at full

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power. The sections are rinsed in PBS and then incubated with a mixture of two primary antibodies for ~30min at room temperature, using appropriate dilutions that have been determined by titration. The pair of primary antibodies are either from different species or of differing Ig isotypes/subclasses. They are washed in TBS and incubated for 1 hr in the dark in secondary antibodies, one conjugated to fluorescein isothiocyanate (FITC) and the other to Texas Red. This is followed by washing in TBS and then counterstaining for 30 sec with DAPI (1 mg/ml of an aqueous solution diluted 1:100 in absolute ethanol). The sections are rinsed in tap water and mounted in antifading medium (DAKO). The slides should be kept at 4°C in the dark when not viewed immediately.

MICROWAVE HEAT–ASSISTED DOUBLE INDIRECT IMMUNOFLUORESCENCE STAINING
Microwave heating is useful for double indirect immunofluorescence staining, provided excessive heating is avoided. Moderate microwaving neither elutes antibodies nor leads to detectable loss of fluorescence and prevents their reactions with subsequently applied reagents. Prolonged staining will elute the antibodies. Moderate heating applied in between the first and second staining cycles facilitates double indirect immunofluorescence staining of antigens using primary monoclonal antibodies raised in the same species. In addition, heating inhibits reactions with endogenous immunoglobulins present in extracellular compartments, which substantially reduces background staining but does not block the staining of the plasma cells and/or lymphocytes. The above procedure has been applied for double indirect staining of mouse pancreatic tissue using fluorescein isothiocyanate (FITC)–conjugated antimouse IgG and Texas Red (Tornehave et al., 2000). Although this method has been successfully used by these authors, it may require amendment for other studies. Before commencing the double staining studies, the potential unmasking effect of microwaving on individual antigen-antibody combinations should be tested because repeated cycles of microwaving will progressively elute the fluorescence-labeled antibodies. With immunoenzymatic detection, this concern is irrelevant as long as the enzyme reaction products remain after microwaving.

Procedure
1. The animal is perfused first with 1–2 ml of saline, followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). Postfixation is accomplished overnight at 4°C in the same fixative. Alternatively, the tissue can be fixed by immersion in the same fixative or 10% commercial formalin in the same buffer. 2. For cryostat sectioning, the tissue specimens are cryoprotected in 30% sucrose in 0.1 M phosphate buffer for 12 hr or until they sink to the bottom of the container. They are embedded in O.C.T compound (Miles, Elkhart, IN) and frozen in Nheptane cooled to the temperature of liquid nitrogen. Alternatively, if the antigens are resistant to paraffin embedding, the specimens can be dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin. 3. Sections ( thick) are hydrated and treated with 1 % bovine serum albumin or with 10% serum from the same species from which the second antibody is derived

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4.

5.

6.

7. 8.

9.
10. 11. 12.

13. 14. 15.

(diluted in 0.05 M Tris buffer [pH 7.4] containing 0.15M NaCl: TBS) for 30 min at room temperature. Antisera or monoclonal antibodies are applied by diluting as required in TBS containing 0.25% BSA (TBS-BSA). Monoclonal antibodies are applied for 1 hr at room temperature, while polyclonal antibodies are applied overnight at 4°C. The sections are rinsed three times for 5 min each in TBS containing 1% Triton X-100. The addition of this detergent is optional but may sometimes reduce background staining. Fluorescence-labeled second antibody is applied for 30 min at room temperature by diluting in TBS-BSA as required. Fluorscein isothiocyanate-(FITC)–Texas Red– or aminomethyl coumarin (AMCA)–labeled variants can be used. The sections are rinsed three times for 5 min each in TBS with or without 1% Triton X-100. If this detergent is used, the sections are rinsed three times for 5 min each in TBS without the detergent before microwaving. Place five slides with sections in a plastic jar containing 50 ml of 10 mM sodium citrate buffer (pH 6.0). The jar is placed in a tray containing 1 liter tap water and microwaved at 780 W. Three cycles of microwaving for 5 min each are adequate. More cycles may lead to losses of antibodies from the sections and fewer cycles may cause inefficient blocking of antibody cross-reactivity. The optimal conditions of microwaving differ depending on the antibody-antigen under study. After microwaving, the slides are left in the citrate buffer at room temperature for 20 min. The sections are rinsed in TBS and then incubated in the primary antibody, which may be raised in the same species as those used in step 1 above. This is followed by rinsing three times for 5 min each in TBS with or without 1% Triton X-100 (cf. step 5). A new round of second antibodies labeled with a fluorophore other than that in step 6 is applied. Alternatively, a triple-layer method employing a second layer of biotin-labeled antiimmunoglobulins followed by fluorescence-labeled streptavidin can be used. They are rinsed three times for 5 min each in TBS with or without 1% Triton X-100. If the detergent is used, at least the final rinse should be in TBS without the detergent. The sections are mounted in a suitable antifade medium such as VectaShield (Vector Laboratories, Burlingame, CA). They are observed with a fluorescence microscope equipped with selective filters for the fluorophores used. An example of the double staining is shown in Figure 8.7 (Plate 4B, C, D).

Control Procedures
In addition to conventional staining and absorption controls, the following procedure is recommended. Parallel sections are processed through the above protocol up to step 10. Instead of specific antibody/antiserum, normal serum from the same species is applied. Steps 11–15 are carried out as described above. These control sections must not show the

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fluorescence characteristic of the label introduced in step 12. If they do, microwaving is insufficient and has not completely blocked the free antigen combining sites presented on the second antibodies introduced in step 6. These sites, if not blocked by microwaving, will bind to immunoglobulins present in the normal serum. These immunoglobulins in turn will bind the second round of second antibodies. Thus, all sites marked by the first antibody will fluoresce with a mixed color characteristic of the two fluorophores used in steps 6 and 12, respectively. Alternatively, it is possible that the first round of second antibodies may not have saturated all binding sites on the primary antibodies, leaving these free to bind second antibodies added in the second staining cycle. In any case, successful microwaving blocks cross-reactivity.

Immunoenzymatic Detection
This procedure is also applicable to immunoenzymatic staining as originally described by Lan et al. (1995). However, with immunoenzymatic detection it is often difficult to discern if different antigens reside in the same cellular compartment. On the other hand, with immunofluorescence, problems with interpreting mixed colors are not encountered because selective filters are used for the individual fluorophores (Larsson, 1988; Tornehave et al., 2000).

COMBINED MICROWAVE HEATING AND ULTRASOUND ANTIGEN RETRIEVAL METHOD
The combined heat-induced epitope retrieval (HIER) and sonication-induced epitope retrieval (SIER) methods have been employed for retrieving cyclin D1/bc l-1 epitope in

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mantle cell lymphoma (Brynes et al., 1997). Compared with HIER or SIER method alone, the combined approach produced stronger immunostaining and lower background staining. Sections of formalin-fixed tissues are mounted on glass slides and heated in a slide oven for 1 hr at 56°C. They are deparaffinized with xylene and rehydrated with ethanol to distilled water. The slides are placed in a plastic microwavable rack and immersed in a microwavable staining dish filled with 200 ml of 0.01 M citrate buffer (pH 6.0). Blank slides are added to the rack to maintain a uniform volume in the container. The container is covered at an angle with the lid and heated twice for 5 min per cycle at 800 W in a microwave oven. Distilled water (50ml) is added after the first heating cycle to cool at room at room temperature for 10 min. The buffer (~800 ml) is heated to boiling in the microwave oven and poured into an 80-W ultrasonic cleaner (Bransonic 12, Branson Cleaning Equipment Co., Shelton, CT). The slide rack is sonicated for 1 min and then cooled in buffer in the staining container for an additional 10 min. The sections are treated with Biotek enzyme (Ventana Biotek) for 10 min, followed by blocking of endogenous peroxidase with 3% hydrogen peroxide in buffer for ~8 min. Immunostaining is carried out in an automated immunostainer (TechMate 1000 Equipment Co., Shelton, CT). The slide rack is sonicated for 1 min and then cooled in buffer in the staining container for an additional 10 min. A cocktail (1:1) of two monoclonal anticyclin D1/bc l-1 antibodies were used: P2D11F11 (diluted 1:40) and 5D4 (diluted 1:100); these two antibodies can be obtained from Vecta Laboratories, Inc., Burlingame, CA, and Immunotech, Westbrook, ME, respectively. The avidin-biotin immunoperoxidase detection system employing DAB is used as the chromogen (Ventana Biotek). After counterstaining in dilute Mayer’s hematoxylin, the sections are dehydrated and mounted in Permount.

COMBINED ENZYME DIGESTION AND MICROWAVE HEATING ANTIGEN RETRIEVAL METHOD
In certain cases maximally effective antigen retrieval conditions tend to cause nonspecific staining and/or background staining. This artifact can be avoided by employing a combination of mild heating and enzyme digestion. This approach can also be used for multiple immunostaining, including that of the PCNA (Ezaki, 2000). Tissue specimens are fixed with 4% paraformaldehyde and embedded in paraffin at 60°C for 1 hr. Sections thick) are mounted on gelatin-coated glass slides, deparaffinized, and rehydrated in distilled water. They are treated with 0.005% pepsin for 15 min at 37°C, followed by heating in 0.01 M citrate buffer (pH 6.0) in a microwave oven (300 W) at 80°C for 15 min. The sections are washed in distilled water for 5 min, rinsed in 0.01 M PBS (pH 7.2) for 15 min, and treated with 0.3–1% to quench endogenous peroxidase activity. They are incubated in the primary antibody (PC10, diluted 1:200 in PBS containing 0.2% BSA for PCNA) for 1–2 hr at room temperature. The sections are washed five times for 5 min each with PBS and then incubated in the enzyme-conjugated secondary antibody in PBS containing 1% heat-inactivated normal rat serum for 1 hr. Horseradish peroxidase

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reaction is developed for 10–20 min at room temperature in a mixture of 10 mg of DAB in 30 ml of PBS and of 30% If needed, 1 ml of 1% cobalt chloride and 1 ml of 1% can be added to the DAB solution to obtain a darker reaction product and increased staining intensity.

PRESSURE COOKER–EDTA–ASSISTED ANTIGEN RETRIEVAL
Immunohistochemical staining of tonsillitis, gastric adenocarcinomas, and breast carcinomas can be obtained using MIB-1 antibody in conjunction with EDTA-NAOH solution and a pressure cooker (Kim et al., 1999b). EDTA solution is thought to be more effective than other buffers in unmasking the epitopes, presumably because it removes (chelates) tissue-bound calcium ions. Deparaffinized and rehydrated tissue sections on slides are immersed in jars containing 1 mM EDTA-NaOH (pH 8.0), and the jars are placed in boiling distilled water in a stainless steel 6-liter-capacity pressure cooker with an operating pressure of 103 kPa/15 psi. The pressure cooker is sealed and brought to full pressure; the duration of heating is ~3 min. The cooker is depressurized and cooled under running tap water for ~20 min. The sections are treated with and then incubated in the primary antibody at a dilution of 1:50. This is followed by sequential incubation in the biotinylated antimouse antibody and streptavidin-biotin-labeled complex. DAB is used for 5 min as the chromogen, and the sections are lightly counterstained with hematoxylin. Positive controls involve the use of the tissue known to express the antigen under study. Negative controls involve the replacement of the primary antibody with the diluent alone or with a nonimmune serum. Note: A 1 mM EDTA solution is easier to set at pH 8.0 when it is buffered. A sodium or potassium phosphate buffer is suitable at 0.005 mM provided the grade of the reagents is of analytical quality, i.e., the content of divalent metals is typically 0.005% or less.

2-MERCAPTOETHANOL–SODIUMIODOACETATE–ASSISTED ANTIGEN RETRIEVAL
Antigen unmasking on sections of paraffin-embedded tissues can be accomplished by reduction of disulfide bonds by treatment with 2-mercaptoethanol, followed by alkylation with sodium iodoacetate to prevent the bonds from reforming. This method has been used for unmasking a Kunitz protease inhibitory domain epitope of Alzheimer’s amyloid precursor protein in human brain (Campbell et al., 1999). Sections are reduced with a mixture of 0.14 M 2-mercaptoethanol in 0.5 M Tris-HCl (pH 8.0) and 1 mM EDTA for 3 hr in the dark at room temperature. After being washed for 3 min in distilled water, the sections are treated with a mixture of 250 mg/ml iodoacetic acid in 0.1 M NaOH, diluted 1:10 in 0.5 M Tris-HCl (pH 8.0) and 1 mM EDTA for 20 min in the dark. The unmasking of antigen with the above method can be enhanced by microwaving the sections for 7 min in 0.05 Tris-HCl (pH 7.0). Controls include preabsorption of the

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primary antibody with the antigen and substitution of the primary antibody with nonimmune serum from the same species. Endogenous peroxidase activity is quenched by treatment with 0.5% in methanol, immunostaining is carried out using a Vectastain Elite ABC kit, and visualization of the secondary antibody is achieved with DAB enhanced with nickel (Vector Laboratories).

ANTIGEN RETRIEVAL WITH STEAM–EDTA–PROTEASE METHOD
Basal cell–specific monoclonal antibody to high-molecular-weight cytokeratins is routinely used to distinguish benign from malignant prostatic acini (Brawer et al., 1985). This antibody provides evidence of the absence of basal cells in prostatic cancer. However, its variable staining tendency under standard preparatory procedures is not uncommon. In the past, antigen retrieval methods used with this antibody consisted of proteolytic digestion, microwave heating, EDTA, or urea treatment. Recently, a combined steam-EDTA-protease protocol was employed in conjunction with this antibody for enhancing basal cell immunoreactivity in noncancerous prostatic epithelium (Iczkowski et al., 1999). This protocol helps to prevent misinterpretation of histological mimics of cancer by improving immunohistochemical basal cell–specific keratin expression in benign prostatic acini and in prostatic intraepithelial neoplasia. The method increases the percentage and intensity of immunoreactivity in basal cells of benign, atrophic, and hyperplastic acini without introducing background staining, thus improving the diagnostic potential of cytokeratin

Procedure
Sections of formalin-fixed and paraffin-embedded tissues are placed onto silanecoated slides, deparaffinized, and rehydrated. They are placed in 0.1 M EDTA (pH 8.0) and exposed to steam heat for 30 min. The slides are cooled for 5 min, rinsed in tap water, loaded onto the ES Autostainer, and treated with Protease 2 (Ventana) for 8 min. Incubation is carried out in the primary antikeratin (diluted 1:10 in PBS) for 32 min and in biotinylated secondary antibodies followed by streptavidin for 8 min each. The staining is visualized on the instrument using 3-amino-9-ethylcarbazole. Between each step, the slides are rinsed in Tris-buffered saline. The results of this protocol are shown in Figure 8.8.

PICRIC ACID–STEAM AUTOCLAVING–FORMIC ACID–GUANIDINE THIOCYANATE–ASSISTED RETRIEVAL OF PRION PROTEIN
In humans Creutzfeldt-Jakob disease (CJD) is the most common of transmissible spongiform encephalopathies (TSEs), a group of neurodegenerative diseases. The cause of the TSEs is the prion protein. A number of immunohistochemical methods are available for detecting prion depositions in the brains of humans suffering from CJD (Kitamoto et al., 1985; Haywood et al., 1994). However, the retrieval of prion in the tissues fixed with formalin and embedded in paraffin is difficult. Hydrated autoclaving instead of microwave heating is necessary for the retrieval of prion protein. The following sequential

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protocol—saturated picric acid–steam autoclaving–formic acid–guanidine thiocyanate—is effective in retrieving this protein (Van Everbroeck et al., 1999). Monoclonal antibodies 3F4 (Senetk, St. Louis, MO) and F89/160.1.5 are used for immunostaining of prion protein. The monoclonal antibody 3F4 was developed by Kascsack et al. (1987) and shows immunoreactivity with the epitope around AA 112 of the human prion protein. The monoclonal antibody F89/160.1.5 was developed at the U.S. Department of Agriculture against a synthetic peptide representing residues 146–159 of

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the bovine prion protein. The epitope has been mapped to the sequence IHFG. This epitope is also conserved in the human prion protein sequence located at AA 139–142. This antibody shows stronger sensitivity and specificity than those obtained with 3F4 antibody for the human prion protein (Van Everbroeck et al., 1999). The antibody F89/160.1.5 is used at a dilution of 1:20,000 with TBSB (150 mM NaCl, 1% BSA, 50 mM Tris-HCl buffer; pH 7.4) (final concentration is 55 ng/ml). The antibody 3F4 is used at a dilution of 1:2,000 with TBSB (final concentration is 1:25 mg/ml).

Procedure
Brain tissue specimens are treated with 98% formic acid for 1 hr to reduce infectivity and embedded in paraffin. Sections thick) are picked up on 0.1% poly-L-lysine-coated glass slides; Superfrost Plus slides are not recommended because sections tend to be dislodged during multiple treatments. The sections on slides are deparaffinized with xylene and rehydrated with descending concentrations of ethanol. They are treated with 5% picric acid for 15 min at room temperature and then thoroughly washed with tap water. The sections are exposed to 0.3% in methanol for 30 min to block endogenous peroxidase. This is followed by autoclaving for 10 min at 121°C using 10 mM citric acid (pH 6.0) as the recovery buffer. The sections are allowed to cool and then washed in distilled water. The sections are treated with 88% formic acid for 5 min and then washed in distilled water. Finally, precooled (4°C) guanidine thiocyanate (4M) is pipetted onto the sections and incubated for 2 hr at 4°C. The sections are exposed to normal swine serum (1:25) in TBSB (150 mM NaCl, 1% BSA, 50 mM Tris-HCl; pH 7.4) for 30 min to block nonspecific binding sites. They are incubated overnight in a humid chamber at room temperature in the primary monclonal antibody (3F4) (Senetk, St. Louis, MO) and diluted 1:2000 with the buffer. The avidinbiotin complex (ABC) method is used to detect antibody binding. The bound antibody is detected by incubation with the secondary antibody (biotinylated goat antimouse IgG diluted 1:100 in TBSB) for 30 min at room temperature. This is followed by incubation for 1 hr with avidin-biotin-horseradish peroxidase complex, diluted 1:200 in TBSB. As the staining mixture, 0.05% DAB in TBS (150 mM NaCl, 50 mM Tris-HCl; pH 7.6) with 0.002% is used for 5 min. After thorough washing for 10 min in running tap water, the sections are counterstained with Harris’ hematoxylin for 30 sec. They are dehydrated and mounted. The results of this procedure are shown in Figure 8.9. Great care should be taken in handling the tissue to avoid infection. Picric acid is both toxic and explosive. Safety guidelines must be used when working with this reagent. Guanidine thiocyanate is also a biohazardous material.

SIMULTANEOUS DETECTION OF MULTIPLE ANTIGENS
Simultaneous detection of multiple antigens provides a spatial relationship between the antigens of interest and saves time, effort, and tissue specimens. In the standard immunoperoxidase technique, horseradish peroxidase is used to oxidize the colorless chromogen DAB into a brown end-product in the presence of hydrogen peroxide. When nickel chloride is included in the reaction mixture, the final reaction product is black. By

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employing these two reagents sequentially, two nonoverlapping antigens can be localized, one exhibiting brown color and the other black. Simultaneous detection of three antigens within one tissue section became possible by employing an additional peroxidase substrate such as the Vector VIP Substrate kit (Vector Lab, Burlingham, CA) (Pujic et al., 1998). This substrate is oxidized by horseradish peroxidase and yields a rose-colored final reaction product which differs in color from that

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of the DAB-based reaction products. The sequence of application is: nickel-enhanced DAB, DAB, and VIP. Three monoclonal antibodies are applied in sequential immunoperoxidase staining steps, resulting in the deposition of black, brown, and rose stains. Colocalization of mast cell tryptase, neurofilament protein, and CD31 in normal human skin was accomplished using this method (Pujic et al., 1998).

Procedure
Human skin tissue is embedded in OCT, cryostat sections are prepared, mounted on poly-L-lysine-coated slides, and stored at –80°C. The sections are brought to room temperature (22°C), and a grease ring is drawn around the sections to limit the spread of reagents. They are rehydrated for 10 min in PBS (0.1M phosphate buffer, 0.15 M saline, pH 7.4) and treated for 15 min with blocking solution containing 2% normal swine serum and 1% BSA in PBS. The sections are incubated for 1 hr with the primary monoclonal antibody, mouse antihuman mast cell tryptase antibody (DAKO), diluted 1:200 with 1% BSA/PBS. They are washed for 10 min in PBS using magnetic stirring, incubated with biotinylated antimouse antibody for 15 min, and washed in PBS. This is followed by adding avidin-biotin-horseradish peroxidase for 15 min. A Vector DAB Substrate kit is applied to develop the reaction product by using nickel-DAB (5 min developing time) according to the manufacturer's instructions. This step yields a black reaction product at sites of mast cell tryptase. The sections are washed in PBS and treated for 15 min with 0.3% to quench residual peroxidase activity. Sections are then treated for 15 min with the blocking solution as described above. The primary monoclonal antibody, mouse antineurofilament protein antibody (DAKO), diluted 1:200 with 1% BSA/PBS, is added for 1 hr. They are washed with PBS and processed through steps with biotinylated secondary antibody and horseradish peroxidase as outlined above. The peroxidase is visualized using a Vector DAB Substrate kit with DAB for a 5-min reaction time to yield a brown reaction product for neurofilaments. Prior to the third staining sequence, the sections are washed in PBS and treated with quenching solution as described above. The primary monoclonal antibody, mouse antihuman CD31, diluted 1:200 with 1% BSA/PBS, is added for 1 hr, followed by washing in PBS. The sections are processed through steps with biotinylated secondary antibody and horseradish peroxidase as described above. The peroxidase enzyme is developed by using a Vector VIP Substrate kit for 4 min to yield a rose-colored reaction product on labeled endothelium. The sections are washed in PBS, counterstained lightly in Mayer’s acid hematoxylin for 20 sec, and rinsed in tap water before being dehydrated in ethanol, cleared in xylene, and mounted.

USE OF MULTIPLE ANTIBODIES FOR LABELING ANTIGENS
Immunohistochemistry of multiple antigens in monolayer cell cultures during the same experimental conditions is also important in cell biology. Advanced techniques for antibody production combined with sensitive detection systems have facilitated the localization of

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picogram quantities of specific antigens (Zijlstra and Schelling, 1999). Two conventional methods for detecting multiple antigens are (1) individual antibodies against different antigens used for separate cell structures and (2) multiple primary antibodies applied against different antigens in the same cell culture and visualized with separate staining methods. Zijlstra and Schelling (1999) have developed the following simple procedure for detecting and quantifying four individual fibronectin isoforms within a single fibroblast monolayer culture.

Procedure
Cells are cultured in 100-mm (internal diameter) tissue culture dishes, rinsed three times at 37°C with PBS, and fixed with 100% methanol for 20 min at –20°C. The methanol is vacuum-aspirated, and residual methanol is allowed to evaporate. A grid (55 X 60 mm) is formed by placing the culture dish on top of a template and tracing the lines using a PAP-pen (the Binding Site, San Diego, CA). Thus, the surface of the dish is divided into 20 distinct areas (11 X 15 mm). The spacing of the area in these grids allows the use of a multichannel pipette. The PAP-pen lines provide a hydrophobic barrier between each area and prevent horizontal movement of fluids. The culture is incubated in methanol containing 3% for 10 min to quench endogenous peroxidase activity. The cells are rehydrated in 70% ethanol for 2 min, followed by rinsing for 5 min in PBS. Nonspecific binding is blocked by incubating the cells for 1 hr in the blocking buffer (1% BSA in PBS). The four monoclonal antibodies used and their specificity are shown in Table 8.1. Approximately of the primary antibodies, appropriately diluted in blocking buffer, are applied to individual areas of the monolayer. The culture dish is placed inside a humid chamber and incubated for 2 hr at room temperature. The template of the grid is used to determine the location, quantity, and dilution of each antibody. Each area is rinsed with of PBS, followed by three changes for 5 min each with blocking buffer A HRP-conjugated secondary antibody, diluted in blocking buffer, is applied to each area for 1 hr in a humid chamber. The areas are rinsed with of blocking buffer. Approximately of DAB is applied for 5 min, rinsed twice with distilled water, and counterstained with Erlich’s hematoxylin. All washing and blocking solutions are applied with a multichannel pipette and removed by aspiration using a 1-ml syringe attached to a vacuum trap. The immunostaining is documented with a Sony DKC-5000 digital photosystem. In the above method, antigen retrieval is not required.

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ANTIGEN RETRIEVAL IN NEURONAL TISSUE SLICES BEFORE VIBRATOME SECTIONING
Microwave heating causes severe wrinkling and folding of free-floating thick vibratome sections (Evers and Uylings, 1994b). To avoid this problem, microwave heating is applied to slices (5 mm thick) of formaldehyde-fixed tissue before vibratome cutting. The following method is useful for antigen retrieval in nonembedded tissue slices from which vibratome sections are prepared for immunohistochemical staining. The following protocol was employed for antigen retrieval in human brain tissues stored in 4% buffered formaldehyde for as long as 4 years (Evers and Uylings, 1997; Evers et al., 1998). A slice ~5 mm thick is cut from fixed brain tissue and washed for several hours in distilled water to remove excess formaldehyde. The slice is immersed overnight in Trisbuffered saline (TBS, pH 9.0) and then placed in a plastic jar containing ~200 ml of TBS. The jar is placed in a microwave oven for 10–15 min at full power (700 W), divided into two cycles of 5 or 7.5 min to check the fluid level. The temperature is controlled using the temperature probe of the oven. It takes ~3 min to reach a temperature of 90°C. The jar is removed from the oven and allowed to cool for 15 min. The slice is rinsed in TBS (pH 7.6), and vibratome sections are cut, collected in plastic vials, and washed for 1 hr in TBS (pH 7.6). (These sections are relatively thick in order to stain whole neurons, including neuronal processes, to distinguish different morphological types.) The sections are immersed in TBS containing 3% hydrogen peroxide and 0.2% Triton X-100 for 30 min to prevent endogenous peroxidase activity. Following a thorough wash in TBS, the sections are placed in TBS containing 5% nonfat dry milk and 0.2% Triton X-100 for 1 hr to prevent nonspecific antibody binding to the tissue proteins. The sections are incubated overnight in the primary monoclonal antibody in a cold room at 4°C. These antibodies (MAP-2, SMI-32, SMI-311, SMI-312, calbindin, and parvalbumin) are diluted 1:1000 to 1:4000 with TBS containing 5% nonfat dried milk and 0.2% Triton X-100. They are thoroughly washed in TBS and then incubated for 1 hr in the secondary antibody. For monoclonal antibodies raised in mice, peroxidase-conjugated rabbit antimouse secondary antibodies can be used. For polyclonal antibodies against calretinin and neuropeptide Y, which are raised in rabbits, goat antirabbit secondary antibodies can be used. After washing in TBS, a tertiary antibody, peroxidase (PAP, 1:1000) can be used. The chromogen used is 0.05% DAB enhanced with 2% nickel-ammonium sulfate.

MICROWAVE HEAT–ASSISTED ANTIGEN RETRIEVAL IN FRESHLY FROZEN BRAIN TISSUE
The use of freshly frozen brain tissues in immunohistochemical studies has certain drawbacks, such as poor preservation of tissue morphology and antigenicity resulting from ice crystal formation. Many antibodies available for immunohistochemistry have not been applied to this type of tissue. Microwave heating at boiling temperature is effective in the rapid retrieval and immunostaining of antigens in freshly frozen tissues, including neural tissues. This technique has been used to enhance staining of the glial fibrillary acidic protein (GFAP) in rat brain tissue using monoclonal anti-GFAP antibody (DeHart et al., 1996). Uniform increased staining of cell bodies and large astrocytic processes occurs in

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both grey and white matter without excessive background staining. If nonuniform staining is observed, it may be due to uneven section thickness or unevenly frozen tissues.

Procedure
Fresh rat brain tissue is immediately immersed in 2-methyl butane at –15 to –25°C for several minutes and stored at –70°C. Sections are cut in the parasagittal plane on a cryostat, which are thaw-mounted onto poly-L-lysine-coated glass slides and stored at –70°C. The sections are dried on a slide warmer at the lowest setting to avoid excessive drying. They are fixed with 4% formaldehyde in PBS (pH 7.5) for 30 min and then rinsed three times for 10 min each in PBS to remove excess fixative. This is followed by treatment with 0.3% hydrogen peroxide in PBS for 30–40 min to quench endogenous peroxidase activity. After being washed in three changes of 5 min each in PBS, the slide is placed in a plastic jar containing 60ml of 10 mM sodium citrate buffer (pH 6.0) and 0.04% Triton X-100. The jar is loosely covered with its screw cap and heated for 5 min in a microwave oven at high power. The buffer starts to boil after ~90 sec. The heating process is interrupted at intervals of 1 min so that the fluid level can be checked and replenished in the jar. The sections must not be allowed to dry. The jar is removed from the oven and allowed to cool to room temperature for 20–30 min. The sections are rinsed in three changes of 5 min each in PBS. The sections are treated with 10% normal horse serum for 3 hr at 4°C to reduce nonspecific binding. After being rinsed three times for 5 min each in PBS, the sections are incubated in the monoclonal anti-GFAP antibody and diluted 1:400 in PBS for 18 hr at 4°C. Following washing three times for 10 min each in PBS, the sections are incubated in biotinylated secondary antibody diluted 1:200 in PBS for 1 hr at room temperature. The peroxidase bridge is completed by treating the sections with an avidin-biotin peroxidase complex solution for 30 min at room temperature. The sections are rinsed twice in PBS, and immunoreactivity is visualized using DAB (10 mg/15 ml) and 0.024% hydrogen peroxide in 50 mM Tris buffer, pH 7.6). After a rinse in distilled water, the sections are dehydrated and cover-slipped.

MICROWAVE HEAT–ASSISTED RAPID IMMUNOSTAINING OF FROZEN SECTIONS
Intraoperative diagnosis requires rapid immunostaining of cryosections. Rapid immunostaining is also helpful in confirming or excluding tumor clearance in resection margins or in detecting micrometastases in sentinel lymph nodes in breast cancer patients. The enhanced polymer one-step staining (EPOS) system allows a rapid one-step immunostaining that can be completed in ~12 min. The EPOS procedure is based on the chemical linking of primary antibodies and horseradish peroxidase to an inert polymer complex (dextran) (Bisgaad et al., 1993). This methodology has been employed for immunostaining of Ki-67, PCNA, cytokeratin, and leukocyte common antigen (Tsutsumi et al., 1995; Richter et al., 1999).

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Procedure
Sections ( thick) of freshly frozen tissues are mounted on silane-coated slides and fixed with 4% buffered formaldehyde (pH 7.0) for 20 sec (Richter et al., 1999). The sections are rinsed in TBS (pH 7.4) for 15 sec, followed by incubation with EPOS antibody for 3 min at 37°C in an incubation chamber. They are rinsed twice for 15 sec each in TBS and then developed with peroxidase-DAB detection kit (Dako) in a microwave oven (500 W) for ~ l min; during microwaving, the slides are cooled by a cold water bath (Werner et al., 1991). After being rinsed in tap water, the sections are counterstained with hematoxylin for 10 sec. They are rinsed in tap water and cover-slipped.

MICROWAVE HEAT–ASSISTED IMMUNOCYTOCHEMISTRY OF THIN CRYOSECTIONS
As is true of formaldehyde-fixed and paraffin-embedded tissue sections for light microscopy, thin cryosections of aldehyde-fixed tissues for electron microscopy also show improved labeling efficiency with microwave heating in some systems. Cryosections can be heated in a microwave oven prior to antibody application. Heating diluted antibodies before their application may also result in improved labeling, but generally this is not true. Typically, protein solutions lose efficiency in the microwave oven. However, the labeling of all types of antigens on cryosections is not improved with microwave heating. Also, the labeling efficiency is affected by the type of fixation and the heating duration. It was demonstrated that optimal labeling, for example, of amylase in thin cryosections of formaldehyde-fixed rat pancreas tissue occurred with microwave heating at 25°C and full power for 2 min (Chicoine and Webster, 1998). In contrast, this duration of heating had no effect on similar sections of the same tissue fixed with glutaraldehyde. On the other hand, thin cryosections of glutaraldehyde-fixed tissues showed increased labeling after microwave heating for 6 min. The duration of heating producing the highest specific signal density in the glutaraldehyde-fixed tissues tends to exert an adverse effect on the signal density of the formaldehyde-fixed tissues. Longer durations of heating are required for the glutaraldehyde-fixed tissue sections because this dialdehyde compared with monoaldehyde formaldehyde introduces more extensive and stronger protein crosslinks. The presence of strong protein crosslinks is a barrier to the accessibility of the antigens to the antibody. Therefore, longer heating is required to break down the protein crosslinks produced by glutaraldehyde. Several possible explanations can be offered to clarify the increased labeling efficiency of antibodies, which may occur because of easy penetration of antibodies into thin cryosections. Antigens are thought to be exposed more readily on cryosections because of the absence of chemical treatments, such as resin embedding and alcohols, after the initial fixation step. Heating of thin cryosections may stabilize the antigens. Cryosections that have been cut from tissue blocks which have been fixed and infiltrated with sucrose are not subjected to additional processing steps (e.g., dehydration with alcohols and embedding in resin), which may wash away soluble antigens such as amylase (Chicoine and Webster, 1998). Heating of thin cyrosections may also unpack the antigen from the surrounding proteins and/or unfold the antigen molecule, facilitating epitope accessibility to the antibody.

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It is known that antibody labeling efficiency over tightly packed antigens is reduced because of steric hindrance.

Procedure
Small pieces of tissue are fixed for 1 hr in a mixture of 4% formaldehyde and 0.5% glutaraldehyde in 100 mM phosphate buffer (pH 7.4) (Chicoine and Webster, 1998). They are infiltrated with 2.3 M sucrose for 24 hr at 4°C, mounted with specimen pins (Leica, Deerfield, IL), and frozen by immersion in liquid nitrogen. The frozen tissue is sectioned at –110°C using an Ultracut E ultramicrotome (Leica) equipped with a diamond knife and an FC4 cryoattachment. Cryosections ~60–80 nm thick are thawed and then mounted on Formvar-carbon-coated grids. A microwave oven equipped with Pello 3420 Load Cooler attachment is set at 25°C and full power. Two beakers, each filled with 500 ml of water, are placed in previously determined hot spots in the microwave oven (see pages 102–103). Grids containing the thin cryosections are floated (section side down) sequentially on small drops of 0.15% glycine and 1% BSA for 15 sec and 5 min, respectively. The reagent drops are placed on a clean disposable plastic surface which has been placed on the cold spot in the oven (an area between the two beakers of water). The local temperature is controlled using the microwave temperature probe immersed in a tube of water placed close to the grids. The sections are floated on small drops of appropriately diluted primary antibody and intermittently microwaved for a total duration of 8 min: 2 min with microwave oven on, 2 min with microwave oven off, 2 min with oven on, and 2 min with oven off. This is followed by washing twice with PBS for 15 sec in a microwave oven. The sections are treated outside the oven for 15 min with protein A–colloidal gold (10 nm) complex diluted 1:20 to 1:50 (determined by spectrophotometry) in PBS containing 1% BSA. They are washed in PBS followed by distilled water and counterstained outside the oven. Alternatively, the sections are microwaved for 6 min before incubation with the primary antibody. The remaining steps are also carried out outside the microwave oven.

PRESSURE COOKER–ASSISTED DETECTION OF APOPTOTIC CELLS
To detect apoptosis in paraffin tissue sections, the TUNEL technique is most commonly used. However, this technique is not specific because it also detects nonspecific DNA degradation in autolysis or necrosis, and DNA breaks during DNA repair, resulting in false-positive staining (Suurmeijer et al., 1999). Alternatively, the apoptotic phenotype can be visualized by immunostaining target proteins cleaved by caspases, because the process of apoptosis is irreversible once the caspase cascade is completely activated. Immunodetection of caspase-cleaved cytokeratin 18 has been accomplished (Caulin et al., 1997). However, this protocol is specific for detecting cytokeratin 18 in apoptotic cells in epithelial tissues or tumors, although immunostaining of cleaved actin filaments is more useful because of its omnipresence in human apoptotic cells. However, whether actin cleavage by caspases is a universal mechanism in apoptosis has not been established as yet.

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A polyclonal antibody (fractin) is specific for an actin fragment generated by caspases during apoptosis. This antibody does not react with intact actin filaments. It is obtained by injecting rabbits with a synthetic peptide (K-YELD) representing the last five amino acids of the C-terminus of the 32-kDa actin fragment produced during apoptosis, residues 240–245 (YELPD) coupled via the K-residue to a carrier protein (purified protein derivative of tuberculin) (Yang et al., 1998). Stock solutions of the antibody are prepared by resuspending of lyophilized rabbit antiserum in distilled water. Suurmeijer et al. (1999) tested three antigen retrieval methods using fractin antibody: microwave heating with 10 mM citrate buffer (pH 6.0), pressure cooking with 1 mM EDTA (pH 8.0), and overnight heating at 70°C with Tris-HCl buffer (pH 9.0). The pressure cooker method yielded the most consistent immunostaining. Light microscopic features of apoptotic cells are nuclear shrinkage and chromatin condensation. Fragmented apoptotic bodies show strong immunostaining (Fig. 8.10/Plate 4E).

IMMUNOHISTOCHEMICAL LOCALIZATION OF PROSTATE-SPECIFIC ANTIGEN
Prostate-specific antigen (PSA) is a chymotrypsinlike serine protease synthesized primarily by the normal, hyperplastic, and malignant male and female prostate. In the male its expression is tightly regulated by androgen through the action of androgen receptor (AR). Upon binding to androgen, AR translocates it into the nucleus and binds to the androgen response elements (AREs) on the PSA promotor, where it interacts with other transcription factors and activates PSA gene transcription. Prostate-specific antigen is currently the most frequently used marker for identifying normal and pathologically altered prostatic tissue in the male and female. At present, there

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is no reason to use the terms Skene’s gland and/or paraurethral ducts and glands for the human female prostate 1999, and Ablin, 2000). Immunohistochemical studies demonstrate that PSA is expressed in the highly specialized, apically superficial layer of male and female secretory cells of the prostate gland, as well as in uroepithelial cells at other sites of the urogenital tract of both sexes. Such studies provide evidence that PSA plays a crucial role in the identification of normal or pathologically altered prostate tissue. In clinical practice, PSA is a valuable marker for diagnosing and monitoring prostate cancer in both sexes. and Ablin (2000) have reviewed the functionalmorphological and some clinical aspects of the normal and pathological female prostate. Prostate-specific antigen is also a serum marker for prostate cancer. The serum PSA is generally proportional to tumor volume and correlates positively with the clinical stage of the disease. Progression of prostate cancer to androgen independence is commonly associated with a rebound of serum PSA. Prostate-specific antigen elevation in hormonerefractory prostate tumors is attributed to mutations and/or amplifications of AR, which broaden its ligand specificity and/or enhance tumor cell’s responsiveness to androgen, respectively. Hormone-refractory prostate cancer is one of the most detrimental diseases affecting men in the United States. In males a range of 1–2 ng/ml in serum is normal in the majority of cases, while values above 3–4 ng/ml are indicative of prostate cancer, benign prostate hyperplasia, or prostatitis. It is noted, however, that exceptions do occur and that up to 40% of individuals with levels <4 ng/ml may have prostate cancer. A healthy female with a normal prostate is characterized by a broad range of serum PSA values from practically unappreciable amounts to the highest reported ones of 0.9 ng/ml. This value is very close to the normal reference range in the male and Ablin, 2000).

IMMUNOHISTOCHEMISTRY
An immunohistochemical examination of PSA using polyclonal antibodies by the peroxidase antiperoxidase (PAP) method and by the technique of biotin-streptavidin-alkaline phosphatase has been successfully carried out et al., 1994). Immunoelectron microscopy in conjunction with the protein A–gold complex can also be used for localizing PSA in human prostate (Sinha et al., 1987). The procedure for immunofluorescence localization of PSA is given below.

Procedure
Paraffin-embedded prostatic tissue sections thick) are deparaffinized and rehydrated (Scorilas et al., 2000). A 5% universal tissue conditioner (Biomeda) is applied for 10 min at room temperature to block any nonspecific binding. The sections are incubated with biotinylated monoclonal mouse antihuman PSA antibody (~2mg/l) (Diagnostic Systems Laboratories [coded 8301]) for 1 hr at 37°C. The sections are stained with for 25 min at 37°C. After each step, the sections are rinsed briefly with 0.5 ml/1 Tween 20 solution. The slides are dried with a stream of cold air, and the resultant fluorescence is observed with a time-resolved fluorescence microscope.

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Chapter 9

Other Applications of Microwave Heating

CARBOHYDRATE ANTIGENS
Molecules other than proteins are also antigenic to various degrees. However, proteins are the most effective antigens because of their size and structural complexity, and almost all proteins larger than 1kDa are antigenic. Complex carbohydrates, especially those bound to proteins or lipids (e.g., glycoproteins and glycolipids) are also of immunological importance. Simple polysaccharides, such as starch and glycogen, are usually not effective antigens because they are rapidly degraded within cells and, in addition, do not form structurally stable epitopes. Lipids are poor antigens because of their wide distribution, relative simplicity, structural instability, and rapid metabolism. However, when linked to proteins or polysaccharides they may function as haptens. Nucleic acids are also poor antigens because of their relative simplicity and flexibility and rapid degradation. Nevertheless, antibodies against DNA and RNA can be produced by stabilizing and linking them to an immunogenic carrier. In fact, several serious human autoimmune diseases, such as systemic lupus erythematosus, are the result of autoantibodies to nucleic acids. The discussion below is limited to carbohydrate antigens. Correlation between molecular alterations and tumor behavior is well established. Tumor behavior is related to the products of changes in cancer-related genes. The study of indirect gene products such as cell surface carbohydrates is important in understanding malignancy because tumor development is usually associated with changes in these carbohydrates. These changes are often divided into alterations related to terminal carbohydrate structures (which include incomplete synthesis and modification of normally existing carbohydrates) and alterations in the carbohydrate core structure. The latter includes chain elongation of both glycolipids and proteins, increased branching of carbohydrates in N-linked glycoproteins, and blocked synthesis of carbohydrates in O-linked mucinlike glycoproteins. The importance of studying such changes becomes obvious considering that the expression of carbohydrate antigens increases in a number of epithelial malignancies, including ovary, lung, bladder, breast, colon, prostate, and gastric carcinomas. In fact, tumor-associated carbohydrate changes are being used in the diagnosis of human cancers, and the expression of some carbohydrate structures is associated with prognosis.
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Carbohydrates such as glycolipids and glycoproteins (including mucins) are extensively expressed on the plasma membrane. They are also present on the membranes of cell organelles such as Golgi apparatus, nuclei, mitochondria, and lysosomes. They function as structural components and are also involved in a variety of cellular functions such as cellular adhesion, recognition and signal transduction, cell matrix interactions, and cell proliferation. Carbohydrate antigens can serve as tumor-associated antigens in carcinomas by virtue of their overexpression or cellular distribution and accessibility. The majority of these carbohydrate antigens are blood group related, belonging either to the A, B, H, or Lewis family, or to the mucin core family (TF, Tn, and sialosyl-Tn). Cellular posttranslational modifications in the maturation of some proteins in both benign and malignant cells require N- and O-linked glycosylation. Changes in glycosylation of both glycolipids and glycoproteins in tumors are well known, and aberrant glycosylation in tumors and tumor-associated carbohydrate antigens has been demonstrated (Hakomori, 1989). It is also known that up-regulated expression or loss of expression of various carbohydrate antigens on the surface of the plasma membrane of cancer cells is associated with a metastatic phenotype. Moreover, such alterations are related to poor patient survival in a number of epithelial malignancies. Carbohydrate antigens are expressed in many organs and tissues, including ovaries; other examples are given below. Sialosyl-Tn antigen is expressed in most of the pancreatic carcinomas, whereas it is completely absent in the normal pancreas (Osaka et al., 1993). Both Tn and sialosyl-Tn antigens have been reported to be present at a higher rate in colorectal carcinomas than in normal colorectal mucosa (Orntoft et al., 1990). Up-regulation of sialylis considered a prognostic parameter in metastatic prostrate cancer (Jørgensen et al., 1995). Sialosyl-Tn antigen expression occurs early in human mammary carcinogenesis and is associated with high nuclear grade and aneuploidy (Cho et al., 1994). Limited expression of sialosyl-Tn has been detected in nonmalignant glandular tissues (e.g., mucinous salivary gland cells, goblet cells of the small intestine and bronchus [Yonezawa et al., 1992; Therkildsen et al., 1994]). However, according to Zimmerman et al. (1999), sialosyl-Tn, as an isolated detection factor, lacks sufficient sensitivity to be of diagnostic value in discriminating malignant from benign mesothelium in body cavity effusions. It should also be noted that sialosyl-Tn expression in a variety of carcinomas is not uniformly detected. Because ovarian carcinoma is the leading cause of death from gynecological cancers, and has been investigated extensively, the involvement of carbohydrate antigens in this cancer is summarized below.

Ovarian Carcinoma
Ovarian epithelial tumors, the most common ovarian malignancy, are usually categorized into four main types: serous, mucinous, endometrioid, and clear cell. The tumors are further classified as benign, of low malignant potential (borderline), or malignant, which are further differentiated into four grades. The histological patterns, especially of malignant tumors, may be a mixture of varying proportions of different histological types. Ovarian carcinoma is often asymptomatic in its early stages. Approximately two-thirds of the patients are diagnosed with stage III and IV disease, when metastatic spread is present within the peritoneal cavity and/or to distant organs. Serous carcinoma is the most serious and common histological subtype of ovarian carcinoma.

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Monoclonal antibodies are available to identify various types of ovarian epithelial tumors through immunohistochemically detecting specific antigens. Antigenic specificities belonging to the O(H) and Lewis blood group families (H-l, H-2, and or the mucin-core family (Tn, sialosyl-Tn and TF) have been studied by using 12 antibodies (Federici et al., 1999). A distinct difference in antigen expression between mucinous and other histological types of ovarian carcinomas (serous and endometrioid) is found. The majority of mucinous tumors express sialosyl-Tn, and antigens strongly and relatively homogeneously, whereas serous and endometrioid tumors rarely express these specificities. In contrast, the latter tumors strongly express and H type 2 antigens. However, according to Tashiro et al. (1994), simultaneous expression of sialosyl-Tn and Tn is observed in both mucinous and serous carcinomas. Such a simultaneous expression is closely connected with malignant change. Sialosyl-Tn and Tn antigens are not expressed in normal ovarian tissues, except for sialosyl-Tn staining of stromal capillaries. Tn is a known cancer-related antigen of the mucin-type polysaccharides. Its presence is the result of incomplete glycosylation. The sialosyl-Tn antigen is formed by the addition of sialic acid to the Tn antigen by 6-sialyltransferase. Immunohistochemical studies have demonstrated that sialosyl-Tn antigen is useful in the histological classification of ovarian carcinomas and in the determination of the malignant potential of such lesions (Ryuko et al., 1993). In contrast to the CA125 antigen, which is a useful tumor marker for nonmucinous adenocarcinomas, sialosyl-Tn is a useful tumor marker for mucinous adenocarcinomas (Kjeldsen et al., 1988). The expression of sialosyl-Tn antigen increases with the transition from benign adenoma to adenocarcinoma. Monoclonal antibody TKH2 (mouse IgGl) specifically recognizes sialosyl-Tn antigen (Fig. 9. 1/Plate 4F). Like Tn antigen, immunohistochemistry of sialosyl-Tn is important because it is a cancer-related antigen. Sialosyl-Tn is not influenced by blood type, menstrual cycle, menopause, pregnancy, or parturition. Recently, Davidson et al. (2000) have evaluated the differences between carbohydrate antigen expression in primary tumors and their respective metastatic lesions, as well as the

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role of these antigens in predicting survival in advanced-stage ovarian carcinomas. This study indicates that sialosyl-Tn, and Tn antigens are widely expressed in primary ovarian carcinomas and their metastases. The diagnostic role of inner-core O-glycans and Lewis family antigens in ovarian carcinomas also has been reported by Dabelsteen (1996). In addition to its role in ovarian cancer, sialosyl-Tn is expressed in many other organs and tissues; some examples are given below. Sialosyl-Tn antigen is expressed in most of the pancreatic carcinomas, whereas it is completely absent in the normal pancreas (Osaka et al., 1993). Both Tn and sialosyl-Tn have been reported to be present in a higher rate in colorectal carcinomas than in normal colorectal mucosa (Orntoft et al., 1990). Up-regulation of is considered a prognostic parameter in metastatic prostate cancer (Jørgensen et al., 1995). Sialosyl-Tn antigen expression occurs early in human mammary carcinogenesis and is associated with high nuclear grade and aneuploidy (Cho et al., 1994). Limited expression of sialosyl-Tn has been detected in nonmalignant glandular tissues (e.g., mucinous salivary gland cells, goblet cells of the small intestine and bronchus) (Yonezawa et al., 1992; Therkildsen et al., 1994). According to Zimmerman et al. (1999), sialosyl-Tn, as an isolated detection factor, lacks sufficient sensitivity to be of diagnostic value in discriminating malignant from benign mesothelium in body cavity effusions. It should be noted that sialosyl-Tn expression in a variety of carcinomas is not uniformly detected.

Microwave Heat–Assisted Carbohydrate Antigen Retrieval
Sections thick) of formalin-fixed and paraffin-embedded ovarian tissue are mounted on silane-coated slides and air-dried for 24hr at 3°C (Davidson et al., 2000). They are deparaffinized, rehydrated, placed in 0.01 M sodium citrate buffer (pH 6.0), and heated twice for 5 min each in a microwave oven. 2H5 antibody (PharMingen, Becton Dickinson, San Jose, CA) is used at a concentration of to detect sialyl antigen. Staining is performed with labeled avidin-biotin. Negative controls consist of the exclusion of the primary antibody, while positive controls consist of carcinomas that have been shown to be immunoreactive for the antigen in earlier studies.

Enzyme Digestion–Assisted Carbohydrate Antigen Retrieval
Both protein antigens and carbohydrate antigens can be retrieved in formalin-fixed and paraffin-embedded tissues using enzyme digestion pretreatment, although the former generally are better retrieved by heat pretreatment. Guhl et al. (1998) have achieved enhanced specificity and intensity of immunogold labeling of sugar moieties (poly a 2, 8 KDN glycotope of megalin) present on O-glycosidically linked oligosaccharides by pretreating the sections with N-glycanase F. This treatment also augments immunogold labeling of certain membrane proteins in thin cryosections at pH 5 to 6. The mechanism responsible for improved detection is thought to be better accessibility of glycotopes to the antibody; glycotopes usually are masked by unrelated large oligosaccharides. The enzyme treatment eliminates steric hindrance by these oligosaccharides. The mechanism of antigen retrieval essentially is based on the depolymerization of

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methylene and polymethylene bridges introduced by formaldehyde fixation. These bridges are known to depolymerize at acidic conditions (pH 5–6).

NUCLEOLAR ORGANIZER–ASSOCIATED REGION PROTEINS
The metaphase nucleolar organizer regions (NORs) are chromosomal segments or loops (rDNA) containing ribosomal genes associated with proteins such as upstream binding factor and RNA polymerase 1. These genes are clustered in 10 loci of the human acrocentric chromosomes 13, 14, 15, 21, and 22. The transcriptional activity of these intranuclear segments plays a pivotal role in the formation of nucleoli, directing the synthesis of both ribosomes and associated proteins. The set of proteins associated with NORs are an acidic, nonhistone type that binds silver ions and that are selectively stained. Silver binds with those rDNA sites that are transcriptionally active or have already been transcribed and still retain residual rRNA non-histone-associated proteins. The NORs stained with silver and the argyrophilic NORassociated proteins are called AgNORs and AgNOR-associated proteins, respectively. The AgNOR proteins appear as distinct black structures under the light microscope. Three different AgNOR staining patterns of metaphase chromosomes in human lymphocytes have been identified (Héliot et al., 2000): pair, sticklike, and unstained structures. Chromosomes 13, 14, and 21 carry predominantly pair or sticklike AgNOR structures, while 15 and 22 carry mainly pair AgNOR structures or remain unstained. Different AgNOR shapes represent both the number of ribosomal genes carried by each chromosome and the differential recruitment of active ribosomal genes in each NOR cluster. In interphase cells, the silver-stained structures are exclusively located within nucleoli (Fig. 9.2). At the ultrastructural level each silver-stained structure corresponds to a fibrillar center with a closely associated dense fibrillar component (Derenzini et al., 1990). Silver-stained NOR-associated proteins play a key role in the control of ribosomal RNA (rRNA) transcription and processing and are considered markers of active ribosomal genes. The close relationship between the rate of cell proliferation and ribosomal biogenesis is well established. In fact, a linear correlation exists between AgNOR counts and the growth fraction in various malignancies, including carcinoma of the breast (Öfner et al., 1996). For example, AgNOR parameters correlate significantly with MIB-1 growth fraction and p53 protein expression (Bànkfalvi et al., 1998). AgNOR expression is markedly higher in cycling (MIB-1 positive) tumor cells than in resting (MIB-1 negative) ones. However, the AgNOR size rather than its number may correlate positively with elevated proliferative status. Cumulative evidence indicates that malignant cells frequently exhibit more AgNOR protein compared with that in benign or normal cells. Pich et al. (2000) have presented a list of 29 tumor types for which AgNOR protein quantity is a prognostic factor. Silver staining of NORs is influenced by the fixative, temperature, and duration of staining. In general, the higher the temperature, the shorter the time required to stain NORs. Prolonged staining results in nonspecific staining, and if staining is excessive, the whole nucleus may appear homogeneously stained with silver. Considering the numerous variables mentioned above and others that may influence NOR stainability, disagreements over interpreting staining results in different laboratories are inevitable. Therefore, standardization of the preparatory protocols is necessary to obtain reproducible results. The standardized silver staining procedure consists of heating the sections in a microwave

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oven, an autoclave, or a pressure cooker. Section staining is carried out in the dark at 37°C using prewarmed solutions. Two methods are available for the quantitative analysis of AgNOR proteins: the counting technique and the morphometric method (Trerè, 2000). The counting technique consists of enumerating each silver-stained dot per cell under the light microscope at a magnification of 100X. The limitation of this technique is that when single AgNOR dots are clustered together or partially overlapped, it becomes subjective and poorly reproducible. Furthermore, the counting technique does not take into consideration the size of each silver-stained dot, which is variable. Clustering of dots and dimensional variability of the dots are common in rapidly proliferating cancer cells. Another disadvantage of this technique is significant interobserver variation. In addition, the technique may fail to demonstrate any prognostic relevance of the AgNOR number to neoplastic diseases, including colorectal carcinoma and breast cancer (Hennigan et al., 1994; Toikkanen and Joensuu, 1993). To circumvent the above limitations, the morphometric method can be used (Rüschoff et al., 1990). It consists of automatic or semiquantitative measurement of the area occupied by the silver-stained structures within the nuclear profile with computer-assisted image analysis (Trerè et al., 1995). This method is faster, more accurate and reproducible, and shows less interobserver variation. Moreover, in contrast to the counting technique, the morphometric method is predictive of patient survival, independent of the clinical stage of the disease (Öfner et al., 1995a,b). The morphometric method can be carried out with a CCD camera mounted on a light microscope and connected to a personal computer equipped with specific morphometric software. For example, Leica Quantimet SOOC image analyzer and processing system can be used. A JVC TK-1280E videocamera, connected to a Leitz Orthoplan light microscope, is used to record the images. QWIN VO1.00 software (Leica) can be used. The area of the nucleus and of each AgNOR, the total area of AgNORs, and the area ratio of AgNORs/ nucleus (AR) are calculated automatically, together with AgNOR length, breadth, perimeter, roundness, and the aspect ratio of each nucleus (Staibano et al., 1998). Values are expressed in micrometers.

Procedure
The following procedure is more suitable for routine application than other methods; as many as 200 specimens can be processed at a time with this procedure. Sections thick) of formalin-fixed and paraffin-embedded tissues are mounted on silane-coated slides. They are deparaffinized with xylene and rehydrated in a series of descending concentrations of ethanol. The sections are immersed in 0.01 M sodium citrate buffer (pH 6.0) in plastic Coplin jars and heated in an autoclave at 120°C for 20min. After the sections have cooled down to room temperature for 20 min, they are incubated in the freshly prepared following silver staining solution for 25 min at room temperature.
2% gelatin in 1% formic acid 25% aqueous silver nitrate 1 part 2 parts

The sections are thoroughly rinsed in deionized water to remove unwanted silver precipitates, dehydrated in a series of ascending concentrations of ethanol, cleared in xylene, and

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mounted. A second set of sections are stained without autoclave pretreatment. The results of this procedure are shown in Figure 9.3.

Nucleolar Size
Hypertrophy of the nucleolus is one of the most distinctive cytological features of cancer cells. Malignant cells usually display a larger nucleolus than do benign cells,

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although nucleolar hypertrophy also occurs in normal proliferating cells. Thus, the value of changes in nucleolar size in tumor pathology has been questioned. However, the relationship between nucleolar function as well as nucleolar size and cell doubling time indicates the importance of the nucleolus in tumor pathology. Also, it has been shown that in cancer cell lines characterized by different proliferation rates, the transcriptional activity of RNA polymerase 1 and the expression of the major nucleolar proteins involved in the control of rRNA transcription and processing (e.g., RNA polymerase 1, nucleolin, fibrillarin, and protein B23) are directly related to nucleolar size and the rapidity of cell proliferation (Derenzini et al., 1998). To evaluate nucleolar size, histological sections are silver-stained for AgNOR proteins. This procedure facilitates clear visualization of nuclear structure and size that can be precisely measured by computer-assisted morphometric analysis (Öfner et al., 1995a). Nucleolar size reliably indicates the rapidity of cell proliferation, inferring the proliferation rate of cancer cells. Higher AgNOR protein values correspond to worse clinical outcomes. Fast-growing tumors have greater rRNA transcriptional activity than slowly growing ones (Derenzini et al., 2000). Therefore, the shorter the cell cycle, the greater is the rRNA transcriptional activity per unit of time and the greater the nucleolar size. This conclusion is logical because proliferating cells must synthesize an adequate ribosomal complement for the daughter cells.

IN SITU HYBRIDIZATION
The in situ hybridization (ISH) technique was introduced in the late 1960s, opening a new era in histology and cell biology (Gall and Pardue, 1969). The technique was originally applied for localizing specific DNA sequences on chromosomes or interphase nuclei. Interphase cytogenetics can be a very useful tool, for example, for bridging the gap between cell culture and histology in tumor cytogenetics. The method is also important for localizing specific mRNA sequences within cells and tissues. It allows the study of chromosomal aberrations in routinely processed tissues. The overall advantage of ISH is that by recognizing specific DNA or RNA sequences in a tissue or a cell, the precise location of a potential or an effective synthesis of a given molecule can be determined. On the other hand, immunocytochemistry demonstrates only the presence of protein molecules after they have been synthesized. In situ hybridization was derived from the techniques of molecular hybridization of nucleic acids that are isolated from a particular cell population or tissue and bound to solid supports. Hybridization of such averaged membrane-bound nucleic acids identifies different classes of DNA (Southern blot) and RNA (Northern blot). However, ISH fills the gap between the detection of a specific sequence and its precise location within the tissue or the cell (Chevalier et al., 1997). Literally hundreds of different hybridizations can be accomplished because ISH is often carried out using semithin or thin sections of single tissue specimen (e.g., surgical biopsy), using light and electron microscopy, respectively. A single specimen, on the other hand, is often insufficient for Northern or Southern blot analysis. Another advantage of ISH is that it allows the establishment of libraries of paraffin- or resin-embedded or frozen tissues. It has been demonstrated, for example, that the hybridization signal can be maintained in frozen sections stored at –70°C with a dessicant for more than 6 years (Wilcox, 1993).

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As in the case of immunocytochemistry/antigen retrieval, a number of factors influence the sensitivity and efficiency of ISH, so they must be applied optimally. The intensity of the ISH signal is affected by the type and concentration of the fixative and the duration of fixation. Dehydration and embedding procedures are also important. All of these procedures must allow the retention of the target of hybridization and/or the hybridized products. The longer the fixation, the more difficult the detection of nucleic acids. The reason is similar to that described earlier for proteins, i.e., strong crosslinking of chromosomal DNA with nuclear proteins. A number of other factors, including the nature of the nucleic acid and its location, the type and efficiency of probe labeling, probe construction and hybridization conditions, and signal detection technique, also contribute to the success of ISH studies. Although the type of nonradioactive probe used does not affect significantly the sensitivity of this technique, the enhancement of signal intensity is influenced by the specific probe used. Another relevant factor is whether enzyme digestion, detergent treatment, or microwave heating is used to enhance ISH detection of nucleic acids. Although enzyme digestion (proteinase K) alone is being employed for enhancing ISH detection of nucleic acids, this approach is not preferred, for it may adversely affect tissue morphology. Moreover, some tissues are resistant to enzymatic digestion. However, proteolytic pretreatment alone is effective for mildly fixed tissues. Denaturing agents such as sodium thiocyanate and Tween 20 have also been used, but the results are capricious and morphological details are not well preserved. However, detergent treatment may have to be used for tissues rich in lipids. In addition, a mild detergent treatment is better for ultrastructural preservation and cell cultures. Microwave pretreatment is the method of choice, for it provides homogeneous hybridization even on large tissue sections. Some evidence indicates that microwave heating is more effective than other types of heating. It is emphasized that the hybridization of labeled probes with the target nucleotides is limited by the ability of the probe to enter the section and by the accessibility and orientation of the target at the surface of the section (Chevalier et al., 1997). In certain cases microwave heating or enzyme digestion alone is insufficient to enhance signal detection. This problem can be avoided by using a combination of microwave heating and enzyme digestion. It was demonstrated, for example, that in comparison with proteinase K digestion or heating alone, short-term (1 min) microwave heating followed by enzyme digestion significantly enhanced the detection of apoptotic cells as well as the staining intensity of the labeled nuclei by the T&T-mediated nick end–labeling technique (TUNEL) (Sträter et al., 1995). The TUNEL method is employed for in situ detection of DNA fragmentation and, thus, of apoptotic cells (Gavrieli et al., 1992). Various mechanisms responsible for the effectiveness of the above-mentioned combined technique have been suggested. Heating may increase the accessibility of proteins to protease attack by stimulating the diffusion and/or enhancing the reaction rates. Microwave heating is known to rapidly hydrolyze proteins and peptides. In addition, microwave pretreatment allows reduction in the effective enzyme concentration as well as duration of enzyme digestion. Proteolytic enzymes also hydrolyze proteins, rendering target nucleic acid more accessible to the probe. Alternatively, microwave heating may directly affect the conformation of the nucleic acid molecule, facilitating denaturation of double-stranded sequences into single strands or unwinding single-stranded structures which may have self-annealed (McQuaid et al., 1990). It has been suggested that high

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temperatures may disrupt the nucleocaspid protein coat of the measles virus, exposing more nucleic acid for in situ hybridization (Shapshak et al., 1985). Another possible explanation is that heating denatures the self-hybrids formed by folding of an mRNA molecule or pseudohybrids formed between neighboring mRNA molecules in tissue sections (Sibony et al., 1995). Heating may loosen these single mRNA strands, enhancing the in situ hybridization efficiency. The role of high temperatures in the breakdown of protein crosslinks introduced by aldehydes has been discussed earlier in this book. The aforementioned combined technique is especially effective in increasing the in situ hybridization signal in archival tissues, the fixation history of which may or may not be known. This protocol also detects low levels of nucleic acids in the tissue, which may not be detectable with heating or enzyme digestion alone. In some cases, the use of medium wattages (e.g., 450 W) is preferred over high-power microwave outputs (e.g., 700 W). This difference has been demonstrated in the ISH for detecting measles virus and chicken anemia virus in formalin-fixed, paraffin-embedded brain tissue (McMahon and McQuaid, 1996). In this study higher power outputs resulted in decreased sensitivity. Although the exact reason for this phenomenon is not known, it is hypothesized that optimal oscillation of dipolar molecules produces optimal thermal effects in tissue sections at medium wattages. Urea (0.01 M), sodium carbonate (0.01 M), magnesium chloride (0.01 M), or distilled water can be used as microwave fluids to obtain similar results in terms of both sensitivity and intensity of the hybridization signal. Alternatively, 10 mM citrate buffer (pH 6.0) can be used as the microwave fluid. The major role of these fluids is to mediate high temperature effects, which is confirmed by the achievement of a good hybridization signal using distilled water. Note that pretreatment conditions must be optimized for every tissue type and for every cell type in a given section.

Radioactive Probes
Radioactively labeled DNA and RNA probes as originally used (Gall and Pardue, 1969) are still widely applied for ISH because of their high sensitivity and strong amplification of autoradiography. Also, radioactive probes enter tissue sections relatively easily. Signal detection can be achieved within weeks with probes, while probes yield autoradiographs within days. The disadvantages of these probes include safety problems, reduced stability of radioactively labeled probes, and long durations of exposure. These and other reasons stimulated interest in the development of nonradioactive probes such as biotin and digoxigenin discussed below.

Nonradioactive Probes
Biotin is a small vitamin molecule ( 244) that binds with high affinity to avidin. Avidin is a larger glycoprotein molecule ( 70,000), mostly distributed in egg whites. This protein has the advantage of conjugating with different markers, including peroxidase, fluorescent dyes, colloidal gold, and ferritin. Because of this property it is

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extensively used in immunocytochemistry to detect biotinylated molecules. However, nonspecific binding of avidin conjugates is not uncommon. Streptavidin obtained from Streptomyces avidin cultures has properties similar to those of avidin and binds biotin with very high affinity. Streptavidin has a neutral pH and is considered superior to avidin with respect to detecting biotinylated ligands. Digoxigenin is a steroid isolated from digitalis plants such as Digitalis purpurea. It is an alternative to biotin for labeling hybridizing probes. Since digoxigenin is present only in the blossoms and leaves of these plants, binding of the antidigoxigenin antibody does not occur in any other biological specimens. It is thought that the efficiency of probe labeling is better with digoxigenin than with biotin, whatever technique is used (nick translation or random priming). This superiority is indicated by the observation that as little as l0pg of digoxigeninated albumin can be visualized in Western blot, whereas the limit of visualization for the biotinylated product is 500pg (Brunet et al., 1994). The avidin-biotin system was developed for detecting antigens at the electron microscope level (Heitzmann and Richards, 1974). Later Heggeness and Ash (1977) proposed the use of this system for fluorescence immunohistochemistry. Guesdon et al. (1979) proposed a variety of labeled avidin-biotin methods which were further supported by Warnke and Levy (1980). The avidin-biotin methods used today are similar to the system described by Hsu et al. (1981). This system is a significant improvement over the previous immunohistochemical techniques. The problem of endogenous biotin is discussed on page 98. Presently, nonradioactive probes, especially biotin or digoxigenin, are favored because they are less hazardous to work with, can be more rapidly developed, and provide better spatial resolution. Thus, introduction of nonradioactive detection systems has made ISH, using formalin-fixed and paraffin-embedded tissues, more accessible for application to molecular cell biology and diagnostic pathology. However, radioactive detection systems are more sensitive than nonradioactive probes, especially oligonucleotide probes used instead of cRNA probes (Sperry et al., 1996). The controversy over the degree to which radioactive probes are more sensitive has not been fully resolved. In any case, microwave pretreatment enhances ISH signal detection of RNA and DNA whether radiolabeled or nonradioactive probes are used; both methods are presented later. Furthermore, a number of approaches is available to increase the sensitivity of the nonradioactive ISH procedures (for a review, see Komminoth and Werner, 1997); some of these approaches are discussed below. Compared with radioactive ISH, nonradioactive ISH requires a 10- to 50-fold higher concentration of probes such as oligonucleotides. However, signal amplification is decreased by increasing probe concentration. Therefore, since nonradioactive probes have limited sensitivity, especially when applied to low-abundance mRNAs, a technique is required for signal amplification. One such technique consists of an optimized protocol for rapid signal amplification based on catalyzed reporter deposition (CARD) that increases the sensitivity of nonradioactive mRNA ISH on the formaldehyde-fixed and paraffinembedded tissues (Speel et al., 1998). This technique facilitates the detection of low-copy mRNAs by ISH (Yang et al., 1999). The CARD approach uses horseradish peroxidase to catalyze the deposition of tyramide molecules that are conjugated to an antihapten immunoglobulin or Streptavidin, which in turn, during the first incubation step, binds to the digoxigenin- or biotin-labeled nucleic acid target. Thus, the number of target sites for the next reaction step is markedly increased.

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During this step, biotin sites on bound tyramine act as additional binding sites for antibiotin enzyme. An additional round of signal amplification can be achieved by using biotinyltyramide and streptavidin conjugated to alkaline phosphatase (Yang et al., 1999). Essentially, the CARD protocol is based on the deposition of haptenized tyramide molecules in the vicinity of hybridized probes catalyzed by horseradish peroxidase. The success of this technique depends on the integrity of target mRNA in sections and the ability of the probe to penetrate the sections and hybridize with mRNAs. Another requirement is an efficient reporter system capable of revealing low numbers of probe-mRNA hybrids per cell accompanied by low background staining. Another approach to improve the resolution and detection sensitivity of the hybridization involves the use of semithin resin sections instead of paraffin sections or frozen sections. The former shows better preservation of cytological details and higher spatial resolution. However, resin sections inhibit probe penetration into the section, limiting probe hybridization to the target sequences/antibodies protruding from the section surface. Such sensitivity and probe penetration difficulties can be circumvented by using the methacrylate embedding–acetone deembedding (MEADE) technique (Warren et al., 1998). These authors successfully localized mRNA and rRNA transcripts in marine bivalves. Other resins such as LR White and Lowicryl K4M do not allow tissue deembedding. Since this method requires longer incubation durations in acetone (12–15min) and also a brief proteolytic digestion of the tissue to optimize intensity of the hybridization signal, such treatments tend to have an adverse effect on cell morphology.

Enhancement of in Situ Hybridization Signal with Microwave Heating (Sibony et al., 1995)
1. Fix human tissues with 4% paraformaldehyde in PBS for 24 hr, embed in paraffin, and cut 4- to sections. 2. Mount sections on silane-treated glass slides. 3. Deparaffinize with three changes of 5min each in xylene, followed by three changes of 100% ethanol, two changes of 95% ethanol, and two changes of 75% ethanol. 4. Rinse in 0.85% NaCl. 5. Place slides in microwave transparent jars containing 0.01 M sodium citrate buffer (pH 6.0), and heat to the boiling point at maximum power (700 W) in a microwave oven equipped with a rotating plate; cover the jars with loosely fitting lids unless 2–3 cm empty space is present above the buffer level in the jar. 6. Allow boiling to continue for 7 min. 7. Check the buffer level, add fresh distilled water if necessary, and again boil for 5 min. 8. Cool the slides to room temperature for 20 min. 9. Rinse for 5 min in PBS (0.145M NaCl and 0.01M sodium phosphate buffer; pH 7.3). 10. Postfix with 4% paraformaldehyde for 20 min. 11. Rinse twice for 5 min each in PBS. 12. Digest with proteinase K for 20 min.

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13. 14. 15. 16. 17.

18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

Rinse in PBS for 5 min. Again postfix with paraformaldehyde for 5 min and rinse in PBS for 5 min. Rinse in 0.85% NaCl. Dehydrate with increasing concentrations of ethanol in 0.3 M ammonium acetate, and air-dry. Hybridize by placing of the following solution for 16hr at 50°C. This is done by lowering the sections in the hybridization mixture with a piece of Parafilm 50% formamide 10% dextran sulfate 1mg/ml salmon sperm DNA 2 × SSC (1 × SSC contains 0.15 mol/liter sodium chloride and 0.015 mol/liter sodium citrate) 70mM DDT Sense- or antisense-radiolabeled riboprobe (concentration adjusted to to cpm/section) Remove Parafilms in 5 × SSC containing 10 mol/liter DTT. Wash with two changes of 30 min each in SSC (five times strength) at room temperature and 50°C, successively. Wash in SSC (double strength) containing 50% formamide and 10 mol/liter DTT for 30min at 55°C. Treat with two changes of 10 min each in NaCl TE (0.5 M NaCl, 10 mM Tris-HCl [pH 7.5], and 5 mM EDTA). To reduce the background, remove all single-strand RNA molecules by digestion with RNAse in NaCl for 20 min at 37°C. Treat with NaCl TE for 90 min at 37°C. Treat with 0.1 × SSC for 15 min at room temperature. Dehydrate with increasing concentrations of ethanol in 0.3 M ammonium acetate, and air-dry. Estimate and quantify macroscopically the hybridization signal on BIOMAX (Kodak, Rochester, NY) films after 5 days of exposure. After immersing the slides in liquid photographic emulsion NTB2 (Kodak), expose them in the dark for 2–6 weeks. Develop and fix photographically. Counterstain with toluidine blue.

Procedure for in Situ Hybridization of DNA
Paraffin sections of formalin-fixed tissues are mounted onto silanized glass slides and air-dried at 60°C (Henke and Ayhan, 1994). They are deparaffinized, rehydrated, and air-dried. The slides are placed in a plastic Coplin jar filled with l0mM sodium citrate buffer (pH 6.0) and microwaved (720 W) for 1 min; this time is measured after reaching the boiling point. After treating with 1 M sodium thiocyanate for 10 min at 80°C, the sections are washed with distilled water and then digested with pepsin (3mg/ml in 0.2 N HC1) for 6 min at 37°C. Both the heating and the pepsin digestion durations must be

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adjusted to the respective tissue. Following washing twice for 5 min each in distilled water, the slides are air-dried and treated on a hot plate for 30 min at 80°C. Each section is covered with of freshly prepared hybridization solution: Deionized formamide (65%) 2 × SSC (0.3 M NaCl and 0.03 M sodium citrate) Dextran (10%) Salmon sperm DNA Biotinylated probe DNA Sections are covered with cover-slips, sealed with rubber cement, denatured by heating at 78°C in a water bath for 10 min, and hybridized overnight at 37°C. The coverslips are carefully removed by floating the slides in 2 × SSC, and the sections are washed twice for 10 min each in a mixture of 2 × SSC and 50% formamide at 40°C, and then three times in PBS. Endogenous peroxidase is blocked by incubation in 1% for 15 min. The labeled DNA is detected by incubating the slides in PBS (10.4mM 3.16mM 150mM NaCl, pH 7.6) containing 1.5% normal horse serum for 10 min at 37°C. The fluid is decanted, and a monoclonal mouse antibiotin antibody, diluted 1:100 in PBS, is added for 30 min at 37°C. The sections are washed three times in PBS and then incubated for 15 min at 37°C in a biotinylated goat antimouse antibody (Vector, Burlingame, CA), diluted 1:1000 in PBS containing 1% BSA. After three washes in PBS, the sections are covered with peroxidase-conjugated streptavidin ( in PBS) for 30 min at 37°C. The sections are carefully washed twice in PBS, and DAB (0.5 mg/ml in 0.05 M Tris-HCl buffer, pH 7.6, and 0.03% ) is added as the chromogen. The sections are counterstained with hematoxylin, dehydrated, and mounted. The results of this method are shown in Figure 9.4.

In Situ Hybridization of RNA in Skeletal Tissues
Skeletal tissues are mineralized and so require decalcification to obtain clear morphological details. Decalcifying reagents such as EDTA and HC1 have been traditionally used at room temperature for processing such tissues for in situ hybridization. However, these and other similar agents require long durations of treatment, resulting in damaged cell morphology and reduced hybridization signals. It has been demonstrated that long decalcification, for example with EDTA at room temperature, reduces hybridization signals, while the signals are preserved better after treatment with the same reagent at the same temperature for short-term decalcification (Kaneko et al., 1998). The above-mentioned limitations can be significantly minimized by using microwave heating. It has been shown that decalcification with EDTA in a microwave oven reduces the duration for decalcification, which in turn prevents the reduction of hybridization signals caused by long-term decalcification (Kaneko et al., 1998). Formic acid has also been used in conjunction with microwave heating as a decalcifying agent and has been reported to decalcify faster than EDTA (Callis and Sterchi, 1998). This treatment has not been tested for in situ hybridization. For in situ hybridization, each consecutive microwave decalcification in the presence of 20% EDTA for 2hr at 50°C for 3–4 days is recommended. The rest of the time, the tissues are decalcified in 20% EDTA at room temperature. A temperature lower than 55°C would safely preserve cell morphology.

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Microwave Heating for in Situ Hybridization of mRNA in Plant Tissues
Traditionally, processing of plant tissues for light microscopy required about 1 week. Although procedures are now available that allow paraffin embedding in short durations (Ruzin, 1999), anatomical preservation is less than excellent. To circumvent these problems, Schichnes et al. (1999) introduced an efficient protocol using microwave heating for paraffin embedding and DIG-labeled (digoxigenin-UTP) knotted (kn) mRNA as a probe for hybridizing mRNA from the knotted gene in the meristematic tissues. This approach decreases the durations of fixation from 24 hr to 15 min, dehydration from 73 hr to l0min, and infiltration from 96 hr to 3 hr. This procedure also minimizes the time required for section adhesion to slides as well as completion of staining. Moreover, anatomical preservation is superior, and localization of the mRNA probe is precise. This protocol is detailed below.
Fixation, Dehydration, and Embedding Step Temperature in Microwave Oven (°C) Time (min)

1. Fixation in PFA (4% paraformaldehyde in PBS), repeat twice (cool on ice in between cycles for 1min) 2. NaCl (0.85%) 3. Ethanol (50%) 4. Ethanol (70%) 5. Ethanol (70% + 0.05% safranin O) 6. Ethanol (100%) repeat once 7. Ethanol (50%) + isopropanol (50%) 8. Isopropanol (100%) 9. Isopropanol (50%) + molten paraffin (50%) 10. Molten paraffin 11. Molten paraffin, repeat four times The total duration is 4hr.

37 67 67 67 67 67 77 77 77 67 67

15 1.25 1.25 1.25 1.25 1.25 1.5 1.5 10 10 30

Microwave Treatment

Areas of high microwave flux are checked with a Pelco 36140 microwave bulb array (Ted Pella). Specimens are not placed in areas indicated by illuminated bulbs. Vials containing the specimens are placed in a cold tap water bath (50 ml) that is preheated to the required temperature. The temperature is regulated by placing the microwave temperature probe into a vial of the same solution that is present in the specimen vial. The built-in temperature probe displays the specimen temperature on the oven front panel. The wire that attaches the probe to the oven is submerged in the water to decrease the antenna effect. An additional 400 ml of static water load is placed in the oven at an optimal position determined with the microwave bulb array. This water is changed between every step.
Staining

Tissue sections ( thick) are placed on Probe-on Plus slides and floated on autoclaved water on a hot plate (42°C) for 2–3 min to remove compression. The water is removed with a paper towel, and the slides are placed on a slotted glass staining dish on its side in the microwave oven. To adhere the sections, the slides are heated in a microwave

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oven at 43°C for 30 min. The probe is placed in a drop of water on a slide on top of the slide rack to measure the temperature. The slides are placed in the staining dish which is filled with 1% safranin O, in 1 part methyl cellusolve, 1 part 50% ethanol, 1% (w/v) sodium acetate, and 2% formalin solution, placed in a water bath in the microwave oven, and loosely covered with plastic wrap to prevent splattering; tight-fitting covers must not be used. The temperature probe is inserted into the staining dish through the wrap. The slides are stained for 45 min at 60°C in the oven.

Microwave Heat–Assisted Fluorescence in Situ Hybridization
If distinct hybridization signals from formalin-fixed paraffin-embedded tissue sections using fluorescence in situ hybridization (FISH) are not obtained, the signals can be enhanced with an appropriate heat treatment. Such a treatment enables the FISH analysis of paraffin sections with poor or uncontrollable fixation conditions (including those of archival specimens) or other problems. Intermittent microwave heating of short durations can rectify some of the problems encountered during conventional FISH. The conventional FISH technique takes a long time to complete, whereas the new method in conjunction with intermittent heating can be completed in only 1 hr. Intermittent heating keeps the temperature as uniform as possible and prevents the sample from drying. This approach yields consistent and distinct signals, without fluctuation in intensities and with minimal background noise. In contrast, conventional FISH tends to result in background autofluorescence that masks weak hybridization signals in the nuclei. Two applications of intermittent heating are given below. Intermittent microwave heating has been used for enhancing FISH signals in the paraffin-embedded tissue sections of gastrointestinal neoplasia (see page 458) (Kitayama et al., 2000). The processing of these tumors is known to be particularly difficult because of the presence of necrosis and contamination with inflammatory and normal stroma cells and poor attachment of paraffin sections to the slide. The second example of the use of the intermittent heating is for identifying centromeres in gastric cancer cells (Kitayama et al., 1999). A panel of 17 centromeric specific probes was used for detecting chromosomal instability in these cells. The study of centromeres is important because chromosomal abnormalities are a well-known characteristic of human cancers.
Procedure for Gastrointestinal Neoplasia

Gastric tumor tissue is fixed with 4% neutral formaldehyde for 1 day and embedded in paraffin (Kitayama et al., 2000). Paraffin sections ( thick) are deparaffinized with xylene and rehydrated with ethanol. Centromeric DNA probes and locus-specific identifier probes (c-myc and p53) are available from Vyis Inc. (Downers Grove, IL). The probes are labeled with orange (Cy 3) or green (FITC) using digoxigenin-11-dUTP and nick translation. The sections are placed in 0.01 M citrate buffer (pH 6.0) and heated in a microwave oven for l0 min. This is followed by treatment with 0.2% pepsin in 0.01N HC1 for l0 min at 37°C, and then exposure to 0.1% NP-40/2 × SSC for l0 min at the same temperature.

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The DNA in the sections is denatured by treatment with 70% formamide/2 × SCC for 5 min at 80°C. Ten microliters of the probe solution (hybridization buffer: probe: and distilled water: ) is placed on the slide and coverslipped. The slide is placed in a microwave oven (2.45 GHz, 300 W) and heated for 3 sec at 2-sec intervals for a total of 15 min at 42°C. DAPI II (4,6-diamidine-2-phenylindol) (125 ng/ml) is used for nuclear staining. The sections are promptly observed under a fluorescent microscope equipped with epifluorescence filters and a photometric CCD camera. The captured images are digitized and stored in an image analysis program. The number of signals per cell is counted for a total of 100–200 cell nuclei, and the signal intensities of the different periods of hybridization are simultaneously compared.

Nuclear Fluorescence in Situ Hybridization Signal Using Microwave Heating
To avoid the limitations of enzyme digestion, microwave heating alone can be used for in situ hybridization. Microwave boiling facilitates the probe’s penetration through the tissue to the nucleus. Also, this treatment causes at least partial denaturation of the nuclei, enhancing hybridization. Microwave heating alone has been employed for fluorescence in situ hybridization for chromosomal sequences (Bull and Harnden, 1999). The pretreatment time is reduced to ~ 1 hr. Prostate tissue is fixed overnight with 4.4% formaldehyde containing 1 % NaCl (pH 6.3). Paraffin sections ( thick) are placed on slides which can be stored at room temperature. The sections are deparaffinized, followed by hybridization. The slides are placed in a glass staining jar containing a few antibumping granules (BDH Lab Supplies, Poole, England), filled to the brim with a mixture of 100 mM Tris-HCl buffer and 50 mM EDTA (pH 7.0), and placed at the center of a rotary 800 W microwave oven. Microwave boiling is carried out at full power for 2–3 min. For four cycles of microwave heating, hot fluid (65°C) is used to refill the staining jar as rapidly as possible to avoid drying of the slides. The slides are transferred to 70% ethanol at 4°C and then to 100% ethanol at the same temperature, followed by air-drying. Digoxigenin-labeled chromosome 10 probe (DIOZI; Oncor, Gaithersburg, MD) is used at a final concentration of in hybridization buffer (50% formamide, 10% dextran sulfate, 0.004% Tween 20, and standard saline citrate (SSC) [in 1.5 strength]). A volume of the probe is placed on an 18 × 18-mm coverslip, which is placed onto the slide, sealed with special Vulcanizing Fluid, and placed on a flat-bed thermal cycler. Slides are incubated for 3 min at 94°C and placed in a humidified box for 16 hr at 37°C. Coverslips are removed, and slides are placed in wash buffer (4 × SSC/0.05% Triton X-100) twice for 2 min each. This is followed by a thorough wash in 0.25 × SSC for 5 min at 72°C, transfer to wash buffer at room temperature, and flooding with blocking buffer (0.5% milk powder in wash buffer) for 5 min. The signal is detected for 30 min at 37°C with antidigoxigenin, diluted 1:100 in blocking buffer. Coverslips are gently removed, and slides are rinsed three times for 2 min each in wash buffer at 45°C before mounting in antifade (Vectashield, Vector Lab., Burlingame, CA) containing propidium iodide Slides are viewed on an Axioskop microscope (Carl Zeiss) equipped with a standard camera and appropriate software (PSI, League City, TX).

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MICROWAVE HEAT–ASSISTED POLYMERASE CHAIN REACTION
The polymerase chain reaction (PCR) technology yields a DNA fragment for cloning and is especially useful for cloning cDNAs. This technique uses the enzyme DNA polymerase to make a copy of a defined region of DNA. The region of the DNA we want to amplify is selected by putting in short pieces of DNA primers that hybridize to DNA sequences on either side of the selected region and cause initiation (priming) of DNA synthesis through that region. The copies of both strands of the selected region, as well as the original DNA strands, serve as templates for the next round of amplification. Thus, the amount of the selected DNA region doubles again and again with each cycle. The PCR technique can be used to study DNA from a variety of sources, including archival specimens containing formalin-fixed, paraffin-embedded tissues (Alcock et al., 1999). Microdissection can be performed on sections cut from such tissues that have been processed for conventional immunohistochemistry. Crude DNA extracts, obtained from microdissected specimens by microwave heating, can be added directly to amplification reactions. Analyses using a range of PCR-based techniques, including microsatellite repeat polymorphism analysis at the NM23-H1 locus and sequencing of exon 5, 7, and 8 of the p53 gene, can be performed.

Procedure
Sections of formalin-fixed, paraffin-embedded tissues mounted on slides are deparaffinized with two washes of xylene for 5 min each and rehydrated in a graded series of ethanol. Endogenous peroxidase activity is quenched by treating the sections with a 0.3% solution of in methanol for 20 min. Standard immunohistochemistry is performed with a Vector stain Elite kit (Vector Laboratories, Burlinghame, CA), using primary antibodies against the protein products of the p53 oncogene (DO7; Novocastra, New Castle upon Tyne, UK) and the putative metastasis suppressor NM23-H1 (anti-nm23Hl/NDPK-A; Novocastra). Immunoreactivity is visualized with DAB, followed by counterstaining with hematoxylin. For immunohistochemistry of p53 antigen, the sections are placed in 0.01 M trisodium citrate fluid for 8 min at 800 W and then allowed to cool to 50°C. The temperature during antigen retrieval is not allowed to exceed 85°C. Following immunohistochemistry, the sections are kept covered with deionized water in a humidified chamber until microdissection on a Leitz Laborlux microscope at X100 magnification. Excess water is drained from the slide, and the area surrounding the section is dried carefully. About of 1 × TE buffer (10 mM Tris, 1 mM EDTA) is placed over the section to create a bubble of liquid over the section. Areas of interest are microdissected with a disposable microlance 3 needle attached to a 10-ml syringe, and harvested with a pipette. Using multiple samples from a section require thorough washing with deionized water to prevent cross-contamination of microdissected samples. The microdissected section fragments in the TE buffer are transferred from the slide to a 0.5-ml plastic tube and centrifuged at 12,000 g for 15 min. The supernatant is discarded, and the tissue pellet is resuspended in of mineral oil and heated in a microwave oven at 800 W for 7 min. After resuspension in an aliquot of TE buffer, the sample is added to the master mix for PCR amplification. The details of amplification and sequence analysis are presented by Alcock et al. (1999).

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DETECTION OF ANTIGENS BY FLOW CYTOMETRY
Flow cytometry has been a versatile method for detecting and quantifying cell surface antigens in diagnostic and research fields for many years. Recently, it has also become useful for simultaneous detection of cytoplasmic and nuclear antigens by using optimal fixation and cell permeabilization protocols. Generally, cell fixation and cell membrane permeabilization are mandatory for the detection of intracellular antigens. Fixation can be carried out with an alcohol or an aldehyde, and cell membrane permeabilization can be accomplished by treating the cells with detergents such as Tween 20, Triton X-100, saponin, lysolecithin, or NP-40. Detergents facilitate access of large molecules such as antibodies and DNA fluorochromes to the target antigen after or before the cells have been fixed. Care is required to achieve optimal cell membrane permeabilization. If permeabilization is insufficient, the accessibility of antibodies to the target antigen will be hampered, resulting in underscoring (Lan et al., 1996). If permeabilization is excessive, cell morphology will be damaged, causing a nondiscrimination pattern in cell populations. Consequently, it is difficult to use light scatter signals to gate on the cells for accurate analysis. Since optimal fixation and detergent permeabilization depend on the type of cell and the characteristics of the antigen under study, these parameters are determined by trial and error. Thus, this approach is somewhat tedious and time consuming. Image cytometric quantitation of nuclear immunostaining can be carried out with the CAS 200 image cytometer (Becton Dickinson, San Jose, CA). It is based on differential staining of nuclei for the antigen (e.g., Ki-67 in ductal carcinoma of the breast using microwave heating and MIB-1 antibody) (Bhoola et al., 1999). Immunopositive nuclei are brown, while negative nuclei counterstain as blue with hematoxylin. Using the two CAS 200 sensors, measurements are made at different wave lengths. At 620 nm, both brown and blue absorb, providing a mask of all nuclear material. At 500 nm only brown stain absorbs, allowing the positive nuclei to be measured independently. Comparison with the 620-nm mask gives a percentage of nuclei area stained positively. The image cytometer is standardized by adjusting the light source of the microscope to a predetermined value on an empty field. Then, in control mode and on the negative control slide, the antibody threshold is adjusted using nuclear areas considered negative. After comparing the brown mask with the blue and the brown-stained image seen through the microscope, the slide stained for the antigen is analyzed in the specimen mode. Fifteen high-power fields are analyzed in each case, using random but consecutive fields when possible. Tumor cells are isolated from stroma by using the scene segmentation function, which allows the operator to precisely define portions of the image to be analyzed (Bhoola et al., 1999). Computer-generated histograms show the percentage of positive nuclear area on the vertical axis and nuclear optical density on the horizontal axis.

Microwave Heat–Assisted Flow Cytometry
The limitations of the application of conventional detergents mentioned above can be circumvented by replacing this approach with cell membrane permeabilization by microwave heating. Improved detection of intracellular antigens can be obtained with microwave heating used in combination with flow cytometry. This approach yields histogram patterns that show clear discrimination between intact cells and cell debris (Fig. 9.5).

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Accurate flow cytometric measurements depend on clear discrimination between intact cells and cellular debris. Both cytoplasmic (bcl-2, CD68, lipocortin-1, muscle actin, and desmin) and nuclear antigens (p53, PCNA, Ki-67) have been simultaneously detected with this combined protocol (Lan et al., 1996; Millard et al., 1998). It has been mentioned earlier in this volume that microwave heating facilitates antibody access to target antigens by breaking down protein crosslinks introduced by formaldehyde during fixation. Microwave heating also preserves cell morphology satisfactorily. Other advantages of this method are easy reproducibility and simultaneous detectability of a number of intracellular antigens. Thus, it has more general application than that of other methods. Although conventional detergent permeabilization can retrieve certain antigens (Teague and El-Naggar, 1994), some other antigens are not detectable. It means that this approach is limited in its application in that each of the detergents allows detection of a limited type of intracellular antigen. According to Millard et al. (1998), improved detection of intracellular as well as surface antigens can be accomplished with flow cytometry using two commercially available chemical reagents, the ORTHOPermeaFix (OPF) and FIX&PERM Cell Permeabilization Kit (F&P). OPF is an aldehyde proprietary mixture of reagents that is commercially available (ORTHO Diagnostic Systems, Raritan, NJ). After fixation of cells with OPF, immunostaining and flow cytometric analysis can be delayed, if necessary, for at least 1 week. Gentle fixation with this reagent can be accomplished in 45 min to 24 hr without any adverse effects (Pizzolo et al., 1994). This property has a practical advantage in that smaller laboratories can collect a patient’s samples to be subsequently sent to a wellequipped laboratory for investigation. Such arrangements also facilitate international collaboration.

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It is thought that the microwave heating method is comparable to the OPF and F&P protocols, though the former method is inexpensive, which is important because in a diagnostic laboratory cost is a consideration in choosing a particular method. Moreover, the exact ingredients of OPF and F&P are not known. It should be noted, however, that unlike these commercial reagents, microwave heating may damage or cleave sensitive cell surface antigens, preventing cell selection on the basis of combined cell membrane and intracellular antigens, but microwaving decreases nonspecific background fluorescence (Millard et al., 1998).

Procedure 1
Cells are washed in PBS and fixed with 2% paraformaldehyde for 30 min at 4°C. The supernatant is discarded after centrifugation (500 g for 5 min at room temperature), and cells are resuspended in 10 ml of 0.01 M sodium citrate buffer (pH 6.0) containing 0.5% bovine serum albumin in an unsealed 50-ml propylene tube. The tube is placed upright in the center of a 1-liter Pyrex glass beaker, which is sealed with polyethylene plastic wrapping. The cell suspension is heated for 30–60 sec in a microwave oven at the maximum power setting (800 W), reaching a temperature between 90 and 100°C. The cells are chilled on ice for 10 min. After centrifugation at 500 g for 5 min, the supernatant is discarded and the cells are washed in PBS. The cells can be filtered through a mesh to remove the aggregated debris and then labeled with monoclonal antibodies for flow cytometry as described below. The cells are incubated in the primary antibody at an appropriate dilution for 30–60min at 4°C or room temperature, depending on the type of cells or antigens. After being washed in PBS, the cells are incubated in a fluorescein isothiocyanate (FITC)–conjugated goat antimouse IgG (or sheep antimouse IgG) for ~30 min at 4°C in the dark. The cells are washed twice in PBS and resuspended in of 1% fetal calf serum or ISOTON II for flow cytometric analysis. The cells are run on a flow cytometer, an EPICS 752 (Coulter Electronics, FL) connected to a CICERO data acquisition system (Cytomation, CO) or FACScan (Bectin Dickinson). An argon ion laser (Coherent, CA) operating at 488 nm is used to illuminate the cells. Forward and right-angle light scatter signals are collected along with FITC fluorescence (measured through a 535-nm bandpass and logarthimic amplification) and PI fluorescence (630 nm long-pass filter) where appropriate. Fluorescence histograms of at least 5,000 counts are generated from a gate set in the forward angle versus 90°C light scatter scattergram. The percentage of positive cells is measured from a cutoff set using an isotype-matched, nonspecific control antibody, while the mean channel fluorescence is measured over the entire distribution. Figure 9.5 shows clear discrimination between intact cells and cell debris after microwave heating. Although the above method is highly recommended, if the expression of immunostaining is weak because of antigen masking and inaccessibility of antigens to antibodies in the aldehyde-fixed and paraffin-embedded tissues, single cell/nuclei can be isolated from archival paraffin-embedded tumors for laser flow cytometry using fluorescentlabeled primary or secondary antibodies. This approach is especially useful for steroid hormones such as estrogen and progesterone, which have nuclear binding sites. The advantage of nuclear isolation is the greater accessibility of immunoreagents to the nuclear proteins compared with that in the nuclei of whole cells.

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Another advantage is the recognition of two subpopulations with low and high staining. Such a heterogeneity of the nuclear antigenic expression (e.g., estrogen) is not seen in the whole cell preparation analyzed by flow cytometry. This heterogeneity is possibly due to improved reactivity and maximum access of the nuclear proteins to the antibodies. Heterogeneity of nuclear protein expression is thought to be due to the variations in the content of nuclear proteins, their cell cycle stage, and proliferation (Sabe et al., 1999). In this respect, different physiological states and differences in size and surface charge of the protein are also important. The nuclear isolation method has been used for processing archival paraffin-embedded mammary tumors for monitoring estrogen expression and aneuploidy (Sabe et al., 1999). These two parameters have important diagnostic and prognostic significance in mammary tumors.

Procedure 2
Approximately sections cut from formalin-fixed and paraffin-embedded tissues (e.g., breast) are treated with 0.05% pepsin (cat. no. P7012, Sigma) in normal saline (pH 1.65) for 1 hr at 40°C (Sabe et al., 1999). The sections are vortexed every 5 min for an additional 30 min. This proteolytic reaction is terminated by adding 5 ml of cold 10% fetal bovine serum (FBS) in ethylene-diaminetetraacetic acid (EDTA). After filtration through nylon mesh, the filtrate is forced through a syringe with a 28-gauge needle and centrifuged at 300g for 7 min. The resulting pellet is resuspended in 1ml of nuclear isolation medium (Hank's PBS with 0.2% FBS, 25 mM HEPES buffer, and 0.6% NP-40) for l0 min at 4°C. The cells and nuclei are aliquoted into polystyrene tubes. Approximately of normal horse serum (Vector Laboratories, Burlingame, CA) is added to block any nonspecific binding. The suspension is incubated with biotinylated antiestrogen monoclonal antibody (1D5, Dako Corp., Carpinteria, CA) at 1:25 dilution for 1 hr at 37°C. Aliquots are stained with of fluorescein isothiocyanate (FITC)–conjugated streptavidin (Dako buffer containing 0.1% Triton X-100). The suspensions are centrifuged and washed twice in 3% FBS/PBS. They are again centrifuged and washed twice in 3% FBS in PBS with 0.1% Triton X-100. The pellets are resuspended and incubated with 0.5 ml of 1% FBS containing propidium iodide ( Calbiochem, San Diego, CA) and RNAse (1 mg/ml) in Hank’s balanced salt solution (without phenol red) for 30 min at 37°C. The samples are analyzed on a Coulter Electronics XL-MCL or a Becton Dickinson FACScan flow cytometer with standard argon ion laser excitation and filter configuration for the FITC/propidium iodide dye combination.

MICROWAVE HEAT–ASSISTED ENZYME-LINKED IMMUNOSORBENT ASSAY
Microwave heat–assisted enzyme-linked immunosorbent assay (ELISA) has been used for measuring anti-glomerular basement membrane (GBM) antibodies in kidney serum (Van Dorp et al., 1991) The presence of GBM antibodies is one of the characteristics of Goodpasture’s syndrome. The application of microwave heating reduces the duration of incubation to assay circulating anti-GBM autoantibodies.

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Antigens in human kidney serum are diluted 1:500 in a solution (0.1M 0.1M and 0.02% Na azide, pH 9.6), and of this dilution is placed in each well of the 96-well microtiter plate. The incubation is carried out for 2 hr at 37°C, and during this period the dilution is exposed to microwave heating for 15 min at a power level of ~ 290W (37.1°C) in the Bio-Rad oven. The power level will differ depending upon the microwave oven used and the position of the microtiter plate in the oven. The plate is positioned on a 5-cm-thick polystyrene platform in the center of the oven to absorb excess microwave energy. The serum is diluted 1:25 in PBS (pH 7.2) containing 0.5% Tween-20 and 1% and of the dilution is placed in each well. It is exposed to microwave heating for 15 min at 41.1°C at the same power level as above. The monoclonal antibody HB43-HRP conjugate is diluted appropriately in PBS containing 0.5% Tween 20 and 1% and 100 ml of the conjugate is placed in each well. It is exposed to microwave heating for 15 min as above. Conventional incubation is carried out by placing 100 ml of the substrate (0.02% 0.04 mg/ml OPD, 0.04M citric acid, and 0.05 M disodium hydrophosphate, pH 5.0) in each well for 20 min at room temperature. The reaction is stopped with of 4 N-sulfuric acid per well. The optical density of the colored solutions is read at 492 nm with a multiphotospectrometer.

MICROWAVE HEAT–ASSISTED SCANNING ELECTRON MICROSCOPY
Like other specimens, bacteria can be processed for scanning electron microscopy (SEM) using microwave heating. Conventional processing of specimens for SEM is carried out in ~4 hr, while they can be prepared in ~1 hr using microwave heating (Fox and Demaree, 1999). Bacterial cells at an early exponential growth phase on polycarbonate membrane filter (Nucleopore Corporation, Pleasanton, CA) can be used. The membrane filter with attached cells is cut into small squares (~50 × 50 mm) and transferred to polypropylene Petri dishes (60 × 15 mm), which are then placed in the cold spots in the microwave oven at power level four (536 W). Previous to this step, using the neon bulb array, cold spot determination has been done. Also, two beakers containing water have been placed in the microwave oven as water loads to absorb microwave energy. The fixation is accomplished by placing ~ 1–2 ml of for 20 sec in the Petri dish. The temperature probe is placed into a blank polypropylene Petri dish during processing, and the temperature is restricted to 37°C to prevent overheating. The filtrate is rinsed three times for 5 min each with 1–2 ml of phosphate buffer. The sample is dehydrated in a microwave oven with 1–2 ml of an ethanol series of increasing concentrations (once in each of 50%, 70%, and 90% ethanol and three times in 100% ethanol). Each dehydration step is carried out for 20 sec in the microwave oven at power level 4 (536 W) with a temperature restriction of 37°C utilizing the temperature probe placed in the blank Petri dish containing ethanol. The filtrate is further dehydrated in 100% hexamethyldisilazine (HMDS) for 20 sec at 37°C in a microwave oven at the same power level utilizing the temperature probe in the blank Petri dish containing HMDS. The filtrate is dried in a conventional oven for 15 min at 60°C. After 15 min all excess HMDS is removed, and the filter with attached cells is allowed

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to continue to dry in the oven. The samples are sputter-coated with gold (1.5 min at 40 mA) and then viewed in a scanning electron microscope. The whole process takes ~ 1 hr. The results of this procedure, as shown in Figure 1.2, are similar to those obtained with standard technique. For additional information on the microwave heat–assisted processing of specimens for scanning electron microscopy, the reader is referred to Demaree (2001).

MICROWAVE HEAT–ASSISTED CONFOCAL SCANNING MICROSCOPY
Confocal laser scanning microscopy can be used in conjunction with microwave heating for examining the three-dimensional structure and cellular interrelationships in sections of paraffin-embedded tissues (Boon and Kok, 1994). Tissues are fixed with Kryofix, a coagulant fixative containing 50% ethyl alcohol and polyethylene glycol (PEG; molecular weight 300) for 90 sec in a microwave oven. The use of thick paraffin sections and fluorescently labeled antibodies is preferred.

MICROWAVE HEAT–ASSISTED CORRELATIVE MICROSCOPY
The choice of tissue processing method is crucial for optimal detection and quantification of target antigens in cells by immunogold light and electron microscopy. The primary criteria for choosing a method are efficient, specific, and reproducible labeling of the antigen and satisfactory preservation of cell morphology. In some cases correlative light and electron microscopy for analyzing immunostaining is desirable. These objectives can be achieved by observing semithin and thin sections of the tissue embedded in water-miscible (e.g., Lowicryl) or water-immiscible (epoxy resins) media. Semithin sections allow a survey of the spatial distribution of the antigen, and thin sections provide subcellular expression of the antigen on consecutive sections. Immunostaining of both semithin and thin epoxy sections can be enhanced by controlled etching of sections with sodium ethoxide (0.6% hydrogen peroxide in 96% ethanol) followed by antigen retrieval in a microwave oven. This procedure was recently used for immunostaining of E-cadherin, and in the human proximal jejunum (Groos et al., 2001). One of the advantages of using an epoxy resin is that, in contrast to cryosections, each tissue block can be repeatedly sectioned for both light and electron microscopy. Treatment with ethoxide permeabilizes the section surface by partial corrosion of the embedding resin. It is essential to determine the optimal concentration of the etching agent and etching duration to obtain sufficient permeability of the section surface for antibody access while avoiding structural damage. It is also necessary to find out optimal heating treatment for unmasking antigens hidden by covalent bonds formed between epoxy resin and biological material during polymerization.

Procedure
Tissue specimens are fixed with 4% formaldehyde (freshly prepared from paraformaldehyde) for 12–18 hr at 4°C, and embedded in Epon (Groos et al., 2001).

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Semithin sections ( thick) are collected on silane-treated slides and air-dried at room temperature. The controlled corrosion of the sections surfaces is obtained by treating them with sodium ethoxide for at least 15 min, then rehydrating them with descending concentrations of ethanol. Antigen retrieval is achieved by transferring the sections in 0.01 M citrate buffer (pH 6.0) and heating three times for 5 min each in a microwave oven at 700 W, followed by cooling to room temperature for 30 min. Nonspecific protein binding is blocked by treating the sections with 5% BSA dissolved in PBS for 30 min at room temperature. After a brief rinse with PBS, the sections are incubated overnight at 4°C with the primary antibody diluted appropriately in PBS containing 1% BSA. They are rinsed in PBS, treated for 1 hr with biotinylated goat antimouse IgG and then treated with peroxidase-labeled streptavidin for 30 min. Peroxidase labeling is visualized with DAB as the chromogen. The sections are rinsed in PBS followed by in rinsing in distilled water, dehydrated in ascending concentrations of ethanol, cleared in xylene, and mounted. Thin sections (~70 nm thick) from the same tissue block are collected on nickel grids. Incubation steps are carried out by floating the grids on drops of each solution, and washing steps are performed by dipping them into the washing solution. Thin sections are corroded by etching with saturated sodium ethoxide diluted to 50% with absolute ethanol for 10 sec. After rehydration and washing in distilled water, antigen retrieval is carried out by heating the sections for 10 min to 95°C and then cooling them to 21 °C at a rate of 0.04°C/sec in a thermocycler. Following washing in three changes of PBS, nonspecific protein binding is blocked by treating the sections with 5% BSA in PBS for 30 min. Incubation with the primary antibody is carried out overnight at 4°C. After application of the 10-nm gold-labeled secondary antibody for 1 hr at a concentration of and subsequent washing in PBS, the immunoreaction is stabilized by treating the sections with 2.5% glutaraldehyde in PBS for 10 min. The sections are counterstained with uranyl acetate and lead citrate. Control sections are processed the same way as the experimental sections, except that either the primary antibody is omitted or is replaced with normal mouse serum in the same concentration as the applied antibody.

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Chapter 10

Cell Proliferating Antigens

Some cell-proliferating antigens that are detected with antigen retrieval methods using frozen or formalin-fixed and paraffin-embedded tissues are discussed below. They include cell nuclear proliferating antigens (Ki-67 and proliferating cell nuclear antigen [PCNA]), p53, estrogen, androgen, and progesterone. Such immunohistochemical studies are important for diagnostic, prognostic, and therapeutic purposes. These studies are in common use to assess the importance of several antigens as prognostic factors in all kinds of malignancies. This approach is more commonly used in pathology laboratories than is analysis of gene mutation at the molecular genetic level, which is cumbersome and time consuming. Before discussing cell nuclear proliferating antigens, it is relevant to briefly explain the cell cycle. Proliferating cells can occupy several functional states besides mitosis. After completing mitosis, the daughter cells enter the Gap 1 phase. The duration of phase varies with the tissue type. Subsequently, cells enter the S (synthetic) phase, where the cell’s genetic material is doubled during DNA synthesis. This phase is followed by a second Gap phase before cells divide again. The durations of these phases in descending order are (8–10 hr), S (6–8 hr), (4–6 hr), and mitosis (30–45 min). Proliferating cells after the phase leave the cell cycle, cease proliferation, differentiate, and eventually die or they enter a resting phase from which they may be recruited back into the cell cycle at a later time.

KI-67 ANTIGEN
Ki-67 is a highly positively charged alkaline nonhistone protein (pI = 9.9) with repetitive elements and a high content of randomly distributed prolines (8.2%) and lysines (11.4%) (Duchrow et al., 1994). It is encoded by a single gene on chromosome 10. The protein is a bimolecular complex of molecular weight 345 and 395 kDa. Ki-67 monoclonal antibody detects two polypeptides of these molecular weights in proliferating cells. Since Ki-67 protein contains many proline–glutamic acid–serine–threonine (PEST) motifs, this protein is capable of being very rapidly catabolized (Rogers et al., 1986). Thus, it has a half-life of only 1–2 hr. Cloning and sequencing of the complete cDNA of Ki-67 antigen have been carried out (Duchrow et al., 1994). The Ki-67 antigen was originally identified by its cell-cycle-related expression and is now considered to be a more specific marker for cell proliferation and cell cycling than is PCNA (Gerdes et al., 1984). This is supported by the observation that ependymal cells,
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which are unable to regenerate, are PCNA-positive, but Ki-67-negative (Sarnat, 1995; Funato et al., 1996). Ki-67 antigen is expressed throughout all phases of the cell cycle ( S, and M), except the quiescent phase. This means that the expression of this antigen is intimately associated with the cell cycle. The topographical distribution of Ki-67 antigen is also cell cycle-dependent. Its expression becomes apparent at the beginning of the phase and accumulates within the nucleus (predominantly in the perinucleolar region) during the late phase. The expression of this antigen increases as the cell cycle progresses, reaches its maximum concentration at mitosis, and markedly diminishes thereafter. In fact, immediately after mitosis its amount is minimal. In the phase Ki-67 antigen is predominantly located in the nucleoli and is particularly evident in cells containing relatively large nucleoli. Treatment with DNase or RNase indicates that the antigen located in the nucleoli is associated with RNA (Szekeres et al., 1995; Benfares et al., 1996). In the later phases of the cell cycle it is also detected throughout the nucleoplasm, being found mostly in the nuclear matrix, where it is associated with DNA. During mitosis, it is present on all chromosomes and appears in a reticulate structure surrounding the metaphase chromosomes (Verheijen et al., 1989). The chromosomal binding of Ki-67 antigen is considered to be due to electrostatic attraction. Because of the short half-life of Ki-67, it is rapidly degraded, resulting in decreased immunostaining during anaphase and telophase. Ki-67 antigen is also sensitive to the nutritional status of cells, declining rapidly after 3 days of nutrient depletion in mitotic cells or becoming undetectable upon nutritional deprivation in lung cancer cells (Verheijen et al., 1989; Tinnemans et al., 1995). However, according to Dong et al. (1997), a greater relative change in Ki-67 expression occurs under conditions where proliferation is inhibited without growth fraction change than under conditions where a significant change in growth fraction does occur. It should be noted that these two observations were made using different cell types. It is apparent from the above discussion that Ki-67 is present in the nucleus of proliferating cells and is an indicator of the growth fraction in tumor cells. It is primarily a DNAbinding protein that plays a crucial role in the maintenance or regulation of cell division. This protein may also function as a matrix for chromosomal DNA or contribute to the condensation of the chromosomes or be involved in breakdown of the nuclear membrane before mitosis (Duchrow et al., 1994). The association of Ki-67 with RNA in the nucleoli and with the DNA with nuclear matrix suggests that the antigen plays a role in transcriptional processes as a structural protein by mediating between nuclear DNA and nucleolar RNA. Ki-67 antigen is a valuable tool for measuring cell growth in human tissues and cell cultures, particularly with respect to the histopathological determination of malignancy. It can provide information on the fraction of actively cycling cells. In certain malignant tumors the number of tumor cells immunohistochemically positive for Ki-67 antigen coincides with estimated tumor proliferation rates (Gerdes et al., 1983). In fact, this antigen is absolutely required for maintaining active cell proliferation. In addition, immunohistochemical evidence indicates that Ki-67 antigen may be associated with neurofibrillary degeneration in Alzheimer’s disease, other neurodegenerative disorders, normal aged brains, and neoplasms such as gangliogliomas (Smith and Lippa, 1995). This antigen possibly plays a role in the production of abnormally phosphorylated tau protein, which leads to the formation of paired helical filaments within susceptible neurons. Considering these

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functions, Ki-67 antigen is expected to be expressed in all active parts of the cell cycle. Unlike PCNA, Ki-67 is not involved in DNA repair. Ki-67 has a much shorter half-life (1–2 hr) than PCNA. Therefore, unlike the latter, the former demonstrates the proliferative stage of the cell rather than the residual evidence of the cell that has passed through the cell cycle stage. It has been demonstrated that normal tissues adjacent to carcinoma are PCNA-positive but Ki-67-negative (Wolf and Dittrich, 1992). On the other hand, in an immunohistochemical study, Ki-67 expression was of no predictive value in squamous cell carcinoma of the head and neck (Roland et al., 1994). A recent study also indicates that the expression of Ki-67 in primary intraoral squamous cell carcinomas of the head and neck is independent of tumor site (Nylander et al., 1997). It has also been demonstrated that Ki-67 antigen staining might contribute to false data on the growth fraction (Ansari et al., 1993). If this problem arises, it can be resolved by employing double labeling with two markers, Ki-67 and statin, for proliferating cells and resting cells, respectively. However, overwhelming evidence has established Ki-67 immunohistochemistry as an important tool for assessing cell proliferation in situ. It should be noted that, like most other antigens, the detectability of Ki-67 is highly influenced by fixation and other preparatory parameters.

Immunohistochemistry
Ki-67 antigen immunohistochemical staining is a simple and reliable procedure for studying tumor proliferative activity in frozen or formalin-fixed, paraffin-embedded tissues, including archival specimens. This antigen can be retrieved on sections of formalinfixed and paraffin-embedded tissues by autoclave treatment (Fig. 10.1) or microwave heating (Fig. 10.2). Both methods are reliable and are presented later. Several antibodies against Ki-67 antigen have been generated (Gerdes et al., 1983, 1984; Key et al., 1993; Kreipe et al., 1993), which are discussed later. The direct diagnostic and independent prognostic value of Ki-67 antigen staining using these antibodies has been well documented. Although Ki-67 antibody has been extensively used and is still being employed, MIB-1 antibody is preferred because it is equally effective in obtaining immunostaining of Ki-67 antigen in frozen or fixed tissues. Fixed specimens show better preservation of cell morphology, allowing clear distinction between positive and negative cells. The above-mentioned advantage also facilitates quantitative immunostaining studies of recently fixed tissues, as well as retrospective examination of archival specimens which have been stored after fixation and embedding. For example, immunostaining of archival specimens (1960–1992) using MIB-l antibody has been carried out for predicting the clinical outcome in patients with acinic cell carcinomas of salivary gland origin (Skalova et al., 1994). This antibody has also been used for immunostaining Ki-67 antigen in human proliferating bone tumor cells using autoclave treatment (Tsuji et al., 1997). This method allows retrieval of this antigen in both formalin-fixed and ethanol-fixed specimens. Note that although immunohistochemistry is contributing significantly to clinical information, in the absence of quantitation, this method is subjective and prone to intra- and interobserver variations. To assure the reproducibility of histopathological results and their correct evaluation, at least an assessment of the percentage of positive cells and both the staining pattern and intensity must be made. If available, computer-assisted image analysis

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can provide an objective and reproducible assessment (Biesterfeld et al., 1995). The presence of Ki-67 antigen can also be determined by flow cytometric methods with improved reproducibility but at the cost of cell morphology details (Steck and El-Naggar, 1994).

Limitations of Immunohistochemistry
The labeling index (percentage of positively stained nuclei) is often found to vary between fields within the same tumor specimen because of the heterogeneous distribution of proliferating cells, which can introduce sampling error. Also, the values obtained for the labeling index may vary among laboratories, depending on storage and handling procedures, thus limiting the usefulness of a direct comparison of the labeling index values. In this respect, interobserver and intraobserver detection variations in the same laboratory also cannot be ignored. Also note that the proliferation rate of the tissue depends not only on the number of cells in the cell cycle but also on the time taken to complete a whole cell cycle and on whether cells undergo programmed cell death. Because the Ki-67 labeling index measures only the number of cells that are cycling and gives no indication of the time required for the cell cycle, a tumor might be proliferating rapidly and still may show a low labeling index, or be proliferating slowly but remain in stage and so have a high labeling index. In other words, a tumor with many cells in a cycle can be strongly immunostained using MIB-1 antibody even though the tumor has a slow cell cycle and a low proliferating rate (Jansson and Sun, 1997). In contrast, a tumor with a short cell cycle and high proliferation rate might not be stained since there are few cells in the cycle. The above-mentioned possibility is one of the reasons for lack of association between Ki-67 immunostaining and clinicopathological variables and prognosis, for example, in colorectal carcinoma. For this and other reasons several studies have indicated that Ki-67 staining has very little prognostic value in gastric and colorectal carcinomas (Victorzon et al., 1997; Jansson and Sun, 1997). However, in spite of these potential limitations, the assessment of proliferation using the Ki-67 labeling index provides valuable prognostic information in many tumor types, including lymphomas, gliomas, and breast tumors. Ki-67 labeling index is considered to be a more objective way of predicting malignant transformation than traditional histopathological evaluation alone.

Antibodies
Monoclonal antibodies Ki-67, MIB-1, Ki-S5, and MIB-5 recognize Ki-67 antigens on sections of formalin-fixed and paraffin-embedded tissues. Using these antibodies in conjunction with immunohistochemistry, a rapid and reproducible determination of the growth fraction of a given human cell population can be accomplished. The determination of the growth factor is an objective aid for defining the outcome of an individual tumor case and is particularly useful for selecting appropriate individual tumor therapy. The Ki-67 antibody was obtained in studies aimed at the production of monoclonal antibodies to nuclear antigens specific to Hodgkin and Sternberg-Reed cells (Gerdes et al., 1983). There is a highly significant correlation between the mean value of the growth

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fraction determined with this antibody and the histopathological grade of malignancy. Immunostaining of Ki-67 antigen with Ki-67 antibody can be enhanced by exposing the cells to increasing ionic strength (1.15–1.6 M NaCl) during fixation with paraformaldehyde, suggesting increased accessibility of the antibody to the epitope (Bruno et al., 1992). Note that epitope denaturation increases at higher ionic strengths. This antibody is used mostly on frozen sections, although it can be employed on sections of fixed and paraffin-embedded tissues after treatment with an antigen retrieval method such as microwave heating. However, the immunoreactivity of this antibody tends to be inconsistent because the epitope in fresh specimens may be lost during routine histopathological processing. To circumvent the limitations of Ki-67 antibody, Ki-67 antibody equivalent murine antibodies (MIB-1–3) were generated against bacterially expressed parts of the Ki-67 cDNA containing three 62-base-pair repetitive elements encoding for the Ki-67 epitope (Key et al., 1993). MIB-1 shows affinity with both native Ki-67 antigen and recombinant parts of the antigen. MIB-1 has excellent immunostaining properties for Ki-67 antigen, not only in frozen tissues but also in routinely fixed and paraffin-embedded specimens. In fact, MIB-1 exhibits an immunostaining pattern identical to that of Ki-67 antibody in fresh specimens. This advantage of MIB-1 allows retrospective studies using archival specimens. The versatility of MIB-1 as a marker of Ki-67 antigen in a wide variety of malignant neoplasms is indicated on pages 39 and 239. Ki-S5 is another antibody generated against the Ki-67 antigen to label a formalinresistant epitope in routinely processed tissues (Kreipe et al., 1993). Crude nuclear extracts of the Hodgkin-derived cell line L428 were used for the immunization of mice and the production of this antibody. The immunoreactivity of Ki-S5 antibody is confined to the nuclei of proliferating cells and, unlike Ki-67 antibody, does not cross-react, for instance, with cytoplasmic antigens of epithelial cells (Rudolph et al., 1995). Moreover, Ki-S5 antibody yields identical results in fresh or fixed tissues. Parallel staining of Ki-67 and Ki-S5 antigens using Ki-67 and Ki-S5 antibodies, respectively, yields almost identical results in nonHodgkin’s lymphoma (Kreipe et al., 1993). Retrospective studies relating the proliferative activity to clinical outcome are rendered possible with antibody Ki-S5 using archival specimens that have been fixed and embedded. Ki-S5 antibody is available free on request from the Institute of Pathology, Kiel, Germany. MIB-5 is yet another antibody that recognizes human Ki-67 antigen (Kosco-Vilbois et al., 1997). Ki-Sl is another IgG mouse monoclonal antibody that was generated by immunizing BALB/C mice with crude nuclear extracts from the human lymphoma cell line U937 (Sampson et al., 1992). This antibody recognizes a 160-kDa cell cycle–associated nuclear antigen and can be used on sections of formalin-fixed and paraffin-embedded tissues. It is considered useful for prognostic information in breast carcinoma (Sampson et al., 1992). To my knowledge, the antigen recognized with Ki-S 1 is uncharacterized. Recently, a new monoclonal antibody, MIB-5 (Immunotech, Westbrook, ME), was generated using bacterially expressed parts of the human Ki-67 cDNA (Gerlach et al., 1997). This antibody is equivalent to the prototype antibody Ki-67 but has the additional advantage of being able to react with the rodent-equivalent, cell cycle–related nuclear protein. MIB-5 antibody identifies cycling cells in embryonic and adult rat tissues fixed with formalin and embedded in paraffin using antigen retrieval with a pressure cooker and immunohistochernistry. The antibody is effective in both fresh and formalin-fixed tissues, as well as in archival specimens for retrospective studies. MIB-5 antibody should also be tried in normal and neoplastic human tissues.

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The aforementioned antibodies recognize different epitopes of the Ki-67 antigen with variable survivals during fixation and embedding or differential expression during the cell cycle. These antibodies have different affinities for the recognized epitopes and influence immunostaining patterns (Mauri et al., 1994). It is known, for example, that although morphological and cell cycle distribution of MIB-1 expression is identical to that of Ki-67 antibody, these two antibodies react with different epitopes of the Ki-67 antigen. Since these antibodies are not interchangeable with one another, the cutoff values to define high- and low-proliferating tumors that have been adopted in previous studies with Ki-67 immunostaining on frozen sections cannot be applied with antibodies such as MIB-1 and Ki-S5.

Recent Applications of MIB-1 Antibody
It is well established that advanced stages of tumor progression are characterized by an increased growth fraction within the neoplastic cell population. The presence of a relevant growth fraction is also related to widely accepted prognostic parameters in human malignancies (Özer et al., 1999). The Ki-67/MIB-l index indicates the proliferation rate of tumor cells and thus is a potential prognostic factor. In addition to its prognostic relevance, the index provides information on the response to clinical treatment, based on the analysis of retrospective and prospective clinical studies. Table 10.1 presents recent examples of the usefulness of Ki-67 antigen–MIB-1 antibody complex as a marker of cellular proliferation (growth fraction). However, the usefulness of this antibody is restricted to certain species and is not applicable, for example, to rat tissues (personal communication, K.-H. Wrobel).

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Ki-67 Antigen Retrieval Using Microwave Heating
Tissue specimens are fixed with 4% formalin for 24 hr and embedded in paraffin. Sections ( thick) are mounted on poly-L-lysine–coated slides, deparaffmized in xylene, rehydrated in a descending series of ethanol, and dried for 1hr at 60°C. Endogenous peroxidase activity is blocked with 1% in methanol for 15 min. The slides are placed in a plastic jar filled with 0.01 M sodium citrate buffer (pH 6.0), which is placed in a microwave oven. They are treated at 750 W for three periods of 5 min each. During each heating cycle the buffer level is checked, and the evaporated portion is replaced with distilled water. The jar is removed from the oven and allowed to cool for 20 min at room temperature. Following a brief rinse in PBS (pH 7.4), 10% normal rabbit serum is applied for 20 min to block nonspecific protein immunostaining. The mouse monoclonal antibody MIB-1 (Immunotech, Westbrook, ME) is applied overnight at 4°C in a humidified chamber. For negative controls, the sections are incubated for the same duration in normal serum in place of MIB-1 antibody. After a rinse in PBS, the sections are incubated in horse antimouse biotinylated antibody (Vector Lab., Burlingame, CA), followed by staining with avidin-biotin complex (Vector Elite ABC) for 1 hr. The peroxidase reaction is developed for 1 min with 0.05% DAB (Sigma) as chromogen. The sections are counterstained with Mayer’s hematoxylin for 1–5 min and mounted in Histomount or gelatin-glycerin. The Ki-67 labeling index (percentage of Ki-67 positive cells) can be determined by scoring 500–1,000 cells. The labeling index can be performed by ocular micrometry on a Leitz or any other appropriate light microscope by using a total magnification of 400. Immunohistochemical staining reactivity is regarded as positive when the stained cells occupy more than 5% of the observed field. Multiple fields of a viable tumor should be examined to minimize erroneous ratings caused by a focal or regional distribution of the proliferating cells (Fig. 10.3). For example, renal tumors are composed of three histological components: undifferentiated embryonic cells (blastema) and variably differentiated epithelial and mesenchymal cells (stroma). Only nuclei with unequivocal reactivity should be scored as positive.

Ki-67 Antigen Retrieval Using Autoclave Treatment
Specimens from a giant-cell tumor of bone are fixed either with 10% buffered formalin or 70% ethanol, decalcified with 5% EDTA in 0.1 M cacodylate buffer (pH 7.4) for 7 days and embedded in paraffin (Tsuji et al., 1997). Sections ( thick) are placed on poly-L-lysine–coated slides (Sigma), deparaffinized, and rehydrated. Endogenous peroxidase is blocked with 1 % (Sigma) in methanol for 5 min. After being rinsed with distilled water, the sections are placed in glass Coplin jars containing 10 mM sodium citrate buffer (pH is adjusted to 6.0 with 2 N NaOH) and heated in an autoclave for 5 min at 100°C. Before being removed from the autoclave, the jars are allowed to cool in the autoclave until the temperature has reached 50°C. After the jars have reached a temperature of 30°C, the sections are incubated overnight at 4°C with MIB-1 antibody at a concentration of The sections are treated successively with biotinylated antimouse IgG antibody diluted 1:300 for 30 min, streptavidin-biotinylated peroxidase complex, diluted 1:50

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for 30 min, and solution prior to counterstaining with hematoxylin. In controls, sections are stained with the conventional ABC method without autoclave treatment. Autoclave heating at 120°C for 5 min results in increased staining but may be accompanied by some damage to the cell morphology. Compared with formalin fixation, ethanol fixation results in stronger staining but less than adequate preservation of cell morphology. Both background staining and false-negative staining are absent. Figure 10.1 shows the results of Ki-67 antigen retrieval using autoclaving.

PROLIFERATING CELL NUCLEAR ANTIGEN
Proliferating cell nuclear antigen (PCNA) is so named because of its initial discovery as an autoantigen found in the nuclei of proliferating cells (Miyachi et al., 1978). It was originally detected with serum from patients with systemic lupus erythematosus, which was found to contain an antibody against a nuclear antigen present in proliferating cells. It was subsequently identified as an S-phase protein and named cyclin, but gradually this term has been phased out. Proliferating cell nuclear antigen is a 36-kDa highly evolutionary conserved eukaryotic, acidic protein at both the protein and DNA sequence levels. Crystallographic studies have shown that PCNA can self-associate as a trimer, forming a hexagonal ring with sixfold pseudosymmetry and a central hole (Gulbis et al., 1996). In the center of the trimer is a cavity that is sufficiently large to accommodate duplex DNA. This cavity is lined with

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positively charged helices facilitating interaction with the negatively charged sugarphosphate backbone of DNA (Cox, 1997). The toroidal structure of PCNA has distinct front and back faces that might provide a variety of sites for interaction with other proteins. The loop region between the twin domains of each monomer is a highly immunogenic exposed site that is important for interaction with other proteins. Such functional protein partners are thought to be crucial in regulating the role of PCNA in replication and repair. The aforementioned symmetry could define the directionality of PCNA movement as it slides along DNA. The cell cycle is composed of S, and M phases, in addition to a resting phase during which cells are quiescent or senescent. The expression of PCNA increases at the end of the phase immediately preceding DNA synthesis, reaches a maximum during the S phase, and declines through phase. It accumulates in larger subnuclear clumps in S phase, which represents matrix-associated replication factories (Cox, 1997). This total nuclear PCNA is tenaciously associated with the replication sites and is not removed by detergents or high salt (Bravo and Macdonald-Bravo, 1987). Although PCNA is present throughout the cell cycle, its levels are almost negligible in long-term mitotically quiescent and senescent cells, compared with proliferating cells and increases dramatically during mitosis. Proliferating cell nuclear antigen is involved in DNA replication as well as in DNA repair synthesis. This antigen is required for processive DNA synthesis catalyzed by DNA polymerase delta, which is one of the enzymes vital for DNA replication. Crystallographic studies show that three PCNA molecules, each containing two topologically identical domains, are tightly associated to form a closed ring (Krishna et al., 1994). The dimensions and electrostatic properties of the ring suggest that PCNA encircles duplex DNA, providing a DNA-bound platform for the attachment of the polymerase. Accumulated evidence indicates that PCNA also plays a critical role in the initiation of cell proliferation, and its expression is elevated almost exclusively during the S phase of the cell cycle. However, not all studies support this observation. This antigen, as detected by PC 10 antibody, does not accurately reflect the S-phase fraction in gastric mucosa, as determined by bromodeoxyuridine (BrdU) labeling (Lynch et al., 1994). Available evidence indicates that PCNA is also involved in DNA nucleotide excisionrepair. This role is exemplified by the demonstration that PCNA can be found associated with chromatin at all phases of the cell cycle after ultraviolet irradiation in vitro (Toschi and Bravo, 1988). Recently it was shown that not only DNA polymerase delta but DNA polymerases beta and epsilon are also involved in the base excision repair subpathways (Dianov et al., 1999). In addition, PCNA may be expressed by noncycling cells in vivo which are undergoing DNA repair (Hall et al., 1993).

Immunohistochemistry
Preservation of cell morphology and antigenicity is a prerequisite to reliable immunohistochemistry of PCNA. Optimal fixation is especially important for antigens involved in DNA synthesis and cell proliferation because correct estimation of the proliferating cell fraction is necessary for diagnostic, prognostic, and therapeutic purposes. For example, in malignant tumors and vascular injury (restenosis), the distinction between quiescent cells and cells going through the cell cycle is important because many therapeutic agents are effective only

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against cells that are proliferating (Mintze et al., 1995). It should be noted that markers such as PCNA can identify those cells progressing through parts of the cell cycle but can fail to detect significant numbers of slow or extended proliferating cell cycle populations. Progressively longer fixation with formalin tends to reduce and eventually may destroy antigenicity. However, in most cases, masked antigens can be unmasked by the antigen retrieval method. Figure 10.4 (Plate 5A, B, C, D) shows the difference between optimally fixed and overfixed tissues in the immunoreactivity of PCNA. Generally, buffered formalin (10%) at pH 7.0 is recommended for fixation for 4hr at room temperature. Although zinc formalin was used for 4–8 hr as a fixative for studying PCNA in pig ileum (Mintze et al., 1995), it is not recommended. PCNA retrieval on sections ( thick) of formalin-fixed and paraffin-embedded tissues can be obtained by heating in a hot water bath at 90°C for 2 hr in 0.01 M sodium citrate buffer (pH 6.0). Alternatively, PCNA can be retrieved by microwave heating (700 W) for two cycles of 5 min each with a 1-min interval in tissues fixed for any length of time (see Fig. 10.3). This treatment is also effective whether tissues are fixed with formalin or Bouin’s solution. The PC10 and 19A2 antibodies are preferred over MAB 424 for PCNA immunohistochemistry, and PC 10 is better than 19A2. Table 10.2 shows recent examples of immunohistochemical localization of PCNA antigen in various carcinomas.

Limitations of PCNA Immunohistochemistry
The reliability of PCNA immunostaining has been questioned (Louis et al., 1991; Harrison et al., 1993; Figge et al., 1992). In fact, some studies have ruled out a prognostic significance for PCNA expression. The use of PCNA as a reliable marker of cell proliferation,

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for instance in intraoral squamous cell carcinoma of the head and neck, has been questioned (Nylander et al., 1997). In certain cases, PC 10 antibody–immunoreactive cells may exceed those expected, especially in neoplasia (Hall et al., 1994). Both the preparatory procedures and endogenous factors play a role in the unpredictability of immunohistochemical results of PCNA. A number of factors—including sample size, fixation, specific epitope involved, type and source of antibody and its concentration, quality of immunostaining and type of detection system, selection of microscopic fields, distinguishing immunopositive nuclei from immunonegative ones, and the threshold at which a particular staining is termed positive—are responsible for variations in the assessment results. In addition, because PCNA immunoreactivity varies considerably within a single tumor specimen, an inexperienced observer can easily miss the areas active in tumorigenesis, especially when the rate of proliferation is low (Sallinen et al., 1994). Examples of endogenous factors that may cause lack of reproducibility of PCNA staining are given below. In some organs normal tissues show high levels of PCNA expression that is not associated with proliferation, i.e., nonproliferating cells also express PCNA (Harrison et al., 1993; Hall et al., 1994). This phenomenon is thought to be due to changes in PCNA regulation in association with neoplasia and the effect of growth factors on transcriptional and posttranscriptional processes (Hall et al., 1990). Growth factors can mediate PCNA expression in cells that need not enter the cell cycle. Epidermal growth factor and TGF have been shown to increase PCNA expression in the mouse pancreas, and it has also been demonstrated that tumors can induce PCNA expression in adjacent normal tissues that are not proliferating (Hall et al., 1994). The above-mentioned phenomenon may also be due to the long half-life (~15–20hr) of PCNA. The long half-life allows cells that are no longer in the cell cycle to continue to exhibit PCNA staining (Scott et al., 1991). Using PCNA immunohistochemistry alone it is not always possible to make a definite distinction between actively proliferating cells and cells arrested in the cell cycle. Thus, arrested cells become a confounding factor. The reproducibility of PCNA immunostaining analysis can be improved by computerassisted image analysis (Sallinen et al., 1994). This approach also improves the reproducibility of quantitation among observers. The effect of tumor heterogeneity is minimized through this protocol because large tissue areas can be analyzed. Moreover, compared with visual assessment, computer-assisted analysis is faster. However, even in the computerized

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assessment, interfield variations resulting from tumor heterogeneity are the primary reason for the potential lack of reproducibility of the proliferation analysis. Figure 10.3 shows PCNA heterogeneity in breast carcinoma. Considering the conflicting opinions expressed in the literature, the specificity of PCNA (and Ki-67) as a proliferation marker needs to be reassessed. In light of the above-mentioned limitations, it seems that Ki-67 is a more specific marker for cell proliferation than PCNA. This suggestion is strengthened by the observation that ependymal cells (which are unable to regenerate) are PCNA positive but Ki-67 negative (Funato et al., 1996). In view of the aforementioned and other factors known to influence PC 10 labeling of PCNA, it should not be accepted uncritically as a marker of cell proliferation in sections of paraffin-embedded tissues.

Immunostaining of PCNA on Cryostat Sections
Although the following method is carried out without the typical antigen retrieval step, it is reliable for immunohistochemical detection of PCNA using cryostat sections (Wrobel et al., 1996). Tissues are fixed by vascular perfusion for 15 min with a mixture of 50% methanol and 10% paraformaldehyde in 10 mM phosphate buffer, followed by additional fixation by immersion for 1 hr in the same fixative. They are hydrated sequentially in 50% and 10% methanol, washed in 0.1M phosphate buffer, and passed through a graded series of sucrose solutions (10%, 20%, and 30%). Following immersion in Tissue TEK OCT Compound (Miles, Elkhardt, IN), the specimens are snap-frozen in liquid nitrogen. Cryostat sections ( thick) are mounted on gelatin/chrome-alum-coated slides and air-dried for 30 sec. The remaining incubation steps are carried out in a moist chamber. The preincubation is carried out for 45 min in the blocking buffer containing 0.1M Tris (pH 7.4), 0.15% Thimerosal, 0.8% Triton X-100, 0.8% NaCl, 20% normal goat serum, and 20% fetal calf serum. After rinsing three times for 10 min each in TBS consisting of 0.1 M Tris (pH 7.4), 0.8% NaCl, and 0.0015% Triton X-100, the sections are incubated overnight at room temperature with the primary monoclonal mouse antihuman PCNA/clone PC10 (diluted 1:3000 in PBS) (Oncogene, Uniondale, NY). The sections are rinsed in TBS as above and incubated for 1 hr in the secondary antibody goat antimouse/biotinylated IgG (diluted 1:200 in the blocking buffer) (Jackson, West Grove, PA). Following rinsing in TBS, blocking of endogenous peroxidase is accomplished by treating the sections with 0.002% phenylhydrazine for 10 min and with 10% for 20 min. This is followed by rinsing in TBS and incubation for 1 hr in avidinbiotin peroxidase complex (ABC) (Vector, Burlingam, CA). The sections are rinsed in TBS as above and developed with 0.5 mg/ml DAB in 0.1 M Tris (pH 7.4) containing 0.002% 0.04% and 0.012% They are rinsed in TBS, dehydrated, and mounted. Controls can be carried out by omitting the primary antibody or substituting the primary antiserum with nonimmune serum diluted 1:500 in blocking buffer. The results of this procedure are shown in Figure 10.5.

P53 ANTIGEN
p53 antigen was discovered before the gene, but both the gene and its protein are called p53. The term p53 was originally given to the phosphoprotein of molecular weight

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53 kDa produced by the p53 gene. This 20-kb human gene consisting of 11 exons is located on the short arm of chromosome 17 in region 17p13.1, and its mutation occurs most frequently in exons 5–9. The exon 5–9 region is highly conserved through evolution and is presumably of functional importance. Approximately 95% of the reported p53

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mutations have been found in these exons and their intervening introns. However, p53 mutations outside exons 5–9 have also been found in human tumors (Greenblatt et al., 1994). In many types of tumors one copy of the short arm of chromosome 17 is often lost. Detailed studies of this chromosome have demonstrated that 17p13.1, which maps the p53 gene, is consistently lost in tumors (Baker et al., 1989). Human p53 protein comprises 393 amino acid residues. It was first detected in SV40 transformed cells by virtue of its ability to form a stable complex with the SV40 large T antigen (Lane and Crawford, 1979). Later it was found that many transformed cell lines, including primary human tumor cells from patients with various types of tumors, contained an elevated level of p53, whereas nontransformed cells contained only small amounts of this protein. The genomic organization of this gene exhibits a striking degree of similarity in different species (Furihata et al., 1995). p53 protein contains three main functional domains: an N-terminal acidic transactivation domain, a central DNA-binding core domain, and a C-terminal homooligomerization domain (Fig. 10.6). All three domains are required for efficient binding of p53 to recognition sites within its physiological target genes and for transcriptional activation of these genes. The vast majority of tumor-associated p53 missense mutations occur within the core domain. More than 95% of the alterations in the p53 gene are point mutations that produce the mutant p53 protein, which in most cases has lost its transactivational activity, resulting in loss of tumor suppressor activity. p53 is the most commonly mutated gene in human cancers, and such a gene is involved in the development of at least 50% of clinical tumors (Darnton, 1998).

Wild-Type p53 Protein
Normal p53 gene is a critical controller of normal growth and homeostasis of cells and tissues. It acts as a guardian of the genome by preventing the proliferation of cells with

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damaged nuclear DNA. This is accomplished by the production of normal (wild-type) p53 protein under normal physiological conditions. This protein is expressed at low levels and has a short half-life due to rapid turnover mediated by ubiquitination and proteolysis. Wild-type p53 becomes stabilized and activated in response to a number of stressful stimuli, including exposure of cells to DNA-damaging agents, hypoxia, nucleotide depletion, or oncogene activation. The activation allows this protein to carry out its function as a tumor suppressor through a number of growth-controlling endpoints. These include cell cycle arrest, apoptosis, senescence, differentiation, and antiangiogenesis. It is apparent from the above discussion that the two primary functions of wild-type p53 protein are growth arrest and apoptosis. These and other functions are elicited by regulating transcription of a number of important genes. In fact, the biochemical activity of this protein relies on its ability to bind to specific DNA sequences and to function as a transcription factor. In other words, wild-type p53 protein acts on downstream genes to arrest the cell cycle until the damaged DNA is repaired or to cause apoptosis (programmed cell death). Apoptosis is an additional, normal mechanism for control of cellular numbers. The concentration of wild-type p53 protein rises in cells after DNA damage, causing arrest of the cell cycle in the (the first gap) phase and blockage of the cell cycle into the S (DNA synthesis) phase via p21 protein. This arrest allows time for DNA repair by interaction of wild-type p53 with downstream activators (Darnton, 1998). As stated above, the arrest of the cell cycle is related to the activation of a number of genes, in particular the WAF1/C1P1 gene that encodes p21 protein. The p21 impedes progression along the cell cycle at the transition, regulating cell proliferation and blocking DNA replication (E1-Deiry et al., 1994). This role of p21 is related to its ability to inhibit cyclin-dependent kinases and PCNA. Therefore, cells lacking p21 may fail to arrest the cell cycle in response to DNA damage. It can be logically assumed that lack of p21 is an indicator of tumor aggressiveness and is correlated with p53 positivity because the mutated p53 product is unable to activate its effector (Zlotta et al., 1999). Many cancers show significant association between p53 abnormalities and lack of p21 expression. However, a p21 expression independent from p53 is a common feature in some cancers, such as malignant ovarian epithelial cell (Elbendary et al., 1996). Nevertheless, combined immunohistological evaluation of p53 and p21 expression deserves careful consideration in histopathological diagnosis. p300 protein also functions in the stabilization of p53 and contributes to the p53 transactivation function in the growth arrest response to DNA damage (Yuan et al., 1999). Accumulation of p53 is due to its stabilization rather than its increased transcription. Deficiency of p300 results in increased degradation of p53. The N-terminal domain of p53 interacts with the C-terminal region of p300. Acetylation of the p53 C-terminal domain by p300 stimulates the DNA binding activity of p53.

Mutant p53 Protein
Tumorigenesis usually proceeds through a series of genetic alterations involving oncogenes and tumor-suppressor genes, each potentially resulting in clonal outgrowth of cells through selective growth advantage. The following brief discussion deals only with one of the latter genes, p53. Mutation of this gene results in the synthesis of a mutated p53

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protein with a changed conformation, a longer half-life (increased stability), and disordered function in terms of cellular growth. It means that mutation of this gene leads to the loss of the guardianship of the genome, which in turn allows progression of cells with damaged DNA through the cell cycle. Loss of control of genomic stability is central in the development of cancer. Both the loss of normal p53 function and the acquisition of oncogenic functions by the mutant p53 protein can contribute to tumorigensis. Both activating and inactivating mutations of the p53 gene can contribute to cancer progression. Inactivation of p53 protein is caused by mutations and deletions in the p53 gene or by interactions of the wildtype p53 protein with oncogenic cellular or viral proteins, for instance, in the primary peritoneal carcinoma (Marchenko and Moll, 1997). Indeed, mutations in the p53 gene occur in high frequency in most of the common types of human cancer. For example, chromosome 17 in more than 50% of both squamous cell and adenocarcinomas of the esophagus harbors missense point mutations (Sasano et al., 1992). Such mutations encode altered forms of the p53 protein. Approximately 85% of the mutations are missense mutations, with one amino acid substituted for another and consequent alteration of p53 protein conformation. Thus, the oncogenic potential of p53 depends on the occurrence of a mutation in its coding sequence. Overexpression of p53 protein is common in human malignant tumors. Accumulation of this protein is usually the consequence of point mutations. Immunohistochemical analyses in many kinds of tumors have demonstrated a good correlation between p53 gene mutation and overexpression of p53 protein. Such a correlation has also been detected directly by DNA sequencing (Furihata et al., 1995). This correlation is particularly clear for colorectal and lung carcinomas. It is well established that overexpression of p53 protein plays an important role in the progression of cancer. However, overexpression of this protein in certain types of tumors has been reported without evidence of p53 gene mutations. Nevertheless, nuclear staining of the majority of tumor cells accompanied by the absence of reactivity in surrounding uninvolved tissues or stroma is the most commonly observed pattern characteristic of the presence of a missense p53 mutation.

p73
The p53 gene product is not the only factor that induces cell cycle arrest or programmed cell death (apoptosis). Two other genes, p73 and p63, encode proteins with transactivation, DNA-binding, and tetramerization domains, and they share considerable homology with p53. Like p53, these proteins also induce cell cycle arrest and apoptosis. Each of these proteins is comprised of several isoforms. The p73 protein is a structural and functional homologue of the p53 protein. cAbl, a nonreceptor tyrosine kinase, regulates p73 to induce DNA damage–mediated apoptosis. Under certain conditions such as DNA damage caused by ionizing radiation or an alkylating agent, c-Abl is activated (White and Prives, 1999). The kinase activity of c-Abl is induced, presumably through the action of the stress-induced ataxia telangiectasiamutated (ATM) gene product, a component of the DNA-damage checkpoint (Shafman et al., 1997). The ATM protein is a widely expressed member of the protein kinases family with similarities to phosphatidylinositol 3-kinases.

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c-Abl binds to p73 in cells, interacting through its SH3 domain with the carboxyl terminal homooligomerization domain of p73. cAbl phosphorylates p73 on a tyrosine residue at position 99 both in in vivo and in cells that have been exposed to ionizing radiation (Yuan et al., 1999). Agami et al. (1999) have also reported that p73 is a substrate for the cAbl kinase, and the ability of c-Abl to phosphorylate p73 is markedly increased by As a result, p73 is able to participate in the apoptotic response to DNA damage. The above findings define a proapoptotic signaling pathway involving p73 and c-Abl. Unlike p53, p73 protein levels do not increase following genotoxic stress. Moreover, although c-Abl interacts with p53 in an irradiated cell, it does not phosphorylate p53 but still contributes to radiation-induced arrest by a p53-dependent mechanism (Yuan et al., 1999). Although p73 is related to p53, p53 alone is the tumor suppressor. p73 protein as yet has not been localized immunohistochemically.

Antibodies
A number of monoclonal and polyclonal antibodies to wild-type p53 and mutant p53 antigens are available and are extensively used in clinical and basic research (Table 10.3). The binding sites for these antibodies on p53 molecule have been identified (Fig. 10.6 and Table 10.4). These antibodies have been a major tool in the immunohistochemical detection of p53 antigen, especially in tumor tissues. The antibodies can be used for frozen or paraffin-embedded tissues; many of them can be employed for both types of specimens, particularly when an antigen retrieval method is used (Table 10.2). This method decreases the immunohistochemical detection threshold of these and other antigens. Such detections rely on the accumulation of these antigens, especially mutant p53 antigen. It should be noted that the threshold-lowering method may detect both wild-type p53 (present in small amounts) and mutant p53 (present in large amounts) in certain cancer-bearing tissues. Such a possibility has been reported in the esophageal squamosa epithelium (Mandard,

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1998). It means that wild-type p53 may be associated with mutant p53. This hypothesis remains to be confirmed by molecular analysis such as sequence analysis. These antibodies are directed against different domains or epitopes of p53 protein. The amino acid sequence of this protein is shown in Figure 10.6. These domains are distributed throughout the p53 molecule from the to the COOH-terminal end. Most of these antibodies recognize the linear epitopes located in the amino- or carboxylterminal regions of p53 protein (Legros et al., 1994a). Studies of antibodies in the sera of mice or rabbits hyperimmunized with human p53 have confirmed that most of these antibodies recognize specific epitopes located in these domains (Legros et al., 1994b). In other words, preferential recognition of amino acid residues 1–95 and carboxyl-terminal residues 300–393 by the antibodies exists (Schlichtholz et al., 1992, 1994). These domains of p53 protein are highly exposed and thus readily accessible to antibodies for immunohistochemical detection. The central region of the protein is thought to be buried in the interior of the molecule. However, Legros et al. (1994b) have been able to direct eight antibodies against this region and thus define four new epitopes. To my knowledge, these eight antibodies as yet have not been used for immunohistochemical studies. Some of the monoclonal antibodies mentioned below recognize different p53 molecule conformations. Also, detection of p53 with different antibodies depends on the time of its synthesis. It has been suggested that the p53 epitope for antibody 1620 remains cryptic immediately after synthesis in human keratinocytes and may not be exposed until late in the life of the protein (Spandau, 1994). Furthermore, different conformations of p53 may predominate in different differentiation stages of the cell or tissue. In addition, differentiation-specific cellular proteins and other proteins that may bind to p53 may mask epitopes on p53 at various stages of differentiation. For example, heat shock protein 70 is known to associate with p53 (Hainaut and Milner, 1992). It is hoped that an understanding of the ability of various anti-p53 monoclonal antibodies to recognize different conformations of p53 in cells will aid in the elucidation of the role played by this protein in cell proliferation, cellular aging or senescence, apoptosis, and gene expression (repressing or stimulating). p53 has been implicated in almost all forms of cell growth stimulation and cell growth inhibition. In addition to the information on antibodies given below, consult Tables 10.3 and 10.4 and Figure 10.6 for their characteristics.

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1. Monoclonal antibody DO-1 (Oncogene Science, Uniondale, NY) detects both wild-type and mutant p53 in cell cultures, frozen sections, and paraffin-embedded sections ( et al., 1992). This antibody binds at the 1–45 amino acid site near the N-terminus of the p53 molecule. 2. Monoclonal antibody DO-2 has properties similar to those shown by the DO-1 antibody ( et al., 1992). 3. Monoclonal antibody DO-7 (Novacastra, New Castle, UK) has the same properties as those shown by the DO-1 antibody ( et al., 1992). Presently, DO7 is the most commonly used antibody for detecting p53. 4. Monoclonal antibody DO-11 recognizes two adjacent peptides (38 and 39) in the PEPSCAN series, localizing the minimum epitope to the 10 amino acids, 181 arginine to 190 proline. The epitope for this antibody lies in the central part of the molecule, the conserved domain III at the surface of the mutant p53 antigen. This antibody completely fails to immunoprecipitate p53 in the wild-type conformation et al., 1995). 5. Monoclonal antibody DO-12 reacts only with a single peptide 54 in the series localizing its site to the 15 amino acids 256 threonine to 270 phenylalanine. The peptide for this antibody lies exactly between conserved domains IV and V. Antibodies DO-12 and DO-11 cross-react with human and mouse p53 and recognize epitopes located in the core part of this protein in areas different from those containing the epitopes for DO-12 and DO-11, which are exposed at the surface of p53 protein. None of the epitopes recognized by DO-11, DO-12, and PAb 240 antibodies contains sites that frequently mutate in human tumors. 6. Monoclonal antibody DO-13 reacts with peptides 7 and 8 in the LPENNVLSPL series (epitope on human p53) near the N-terminus. This antibody, like DO-1 antibody, recognizes conformations of both wild-type and mutant p53 antigens and reacts with human p53 but not with murine p53. 7. Monoclonal antibody DO-14 reacts with peptides 13 and 14 in the EDPGPDEAPR series within the N-terminal region. This antibody recognizes conformations of both wild-type and mutant p53 antigens. It reacts with human p53 but not with murine p53. In summary, the epitopes recognized by DO-11, DO-12, DO-13, and DO-14 antibodies are restricted to regions of 10–15 amino acids of human p53. 8. Monoclonal antibody PAb 240 (Oncogene Science, Uniondale, NY) recognizes a linear epitope clearly defined as being within the central core of the mutant p53 molecule (Gannon et al., 1990). The epitope for this antibody is cryptic in the active DNA binding form of wild-type p53 but is exposed at the surface of many mutant p53 proteins as well as of denatured wild-type p53 protein. PAb 240, DO-11, and DO-12 antibodies do not precipitate all of the mutant p53 proteins, suggesting that in some mutant molecules the epitopes remain cryptic. Cryptic epitopes can be exposed by denaturation or through mutations. PAb 240 antibody can be used for wild-type and mutant p53 proteins on fresh or paraffinembedded tissues with the aid of an appropriate antigen retrieval method. Because PAb 240 reacts with a conformational-dependent epitope in the p53 molecule, this antibody has helped define the occurrence of different conformational forms of the p53 protein.

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9. Monoclonal antibody PAb 1801 (Oncogene Science, Uniondale, NY) recognizes the N-terminal epitope that is denaturation-resistant. 10. Monoclonal antibody Bp53-12 (BioGenex, San Ramon, CA). 11. Polyclonal antibody CM-1 (BioGenex, San Ramon, CA) was raised against fulllength human mutant p53 protein (Midgley et al., 1992). 12. Polyclonal antibody R19 (Biotechnology, Santa Cruz, CA) binds near the carboxy-terminus of rat p53 (Uberti et al., 1998).

A combination of antibodies has been used in immunohistochemistry. Resnick et al. (1995) have compared the efficacy of immunostaining of accumulated p53 in fresh-frozen as well as formalin-fixed and paraffin-embedded lung and upper aerodigestive tract carcinomas, using antibodies PAb 1801, DO-7, DO-1, or a 1:1 mixture of PAb 1801 and DO-7 (or DO-1), in conjunction with microwave pretreatment. Although these antibodies used alone yielded good immunostaining, the PAb 1801–DO-7 (or DO-1) mixture showed the staining of the greatest number of cells. The higher sensitivity of the staining achieved with the mixture is related to the numerous binding sites on the p53 molecule available to the two antibodies. In other words, the cumulative binding of two antibodies exceeds the binding of any one antibody. As indicated in Table 10.3, each antibody has affinity for different epitopes. A mixture of monoclonal antibodies BaGS-3 (1:80) and BaGS-5 (1:80) has also been employed for the detection of T and Tn epitopes on breast adenocarcinoma cells (Wang et al., 1998). However, the use of a mixture of antibodies is not in common use.

Examples of Antibody Dilutions
As an example, optimal dilutions of four antibodies commonly used for p53 protein in squamous cell carcinomas are given below (Piffko et al., 1995).
CM1 DO-7 PAb 240 PAb 1801 1:2,000 1:200 1:10 1:40

Note that a wide range of dilutions of the same antibody is used, depending on the tissue type and whether or not an antigen retrieval method is used. For example, DO-7 antibody has been used at a dilution of 1:10 for detecting p53 protein in esophageal carcinoma (Yang et al., 1998), while the same antibody was employed at a dilution of 1:1,000 for detecting this protein in breast cancer tumors (Daidone et al., 1998). There are many similar examples. Substantial differences in the quality and quantity of immunostaining of p53 are found even in the same tissue, depending on the primary antibody and the dilution used.

Immunohistochemistry
Wild-type p53 protein is difficult to detect immunohistochemically in normal cells because of its very short half-life (~20 min) and its presence in minute amounts. However, wild-type p53 protein accumulation can be detected by using antigen retrieval techniques (Dowel and Ogden, 1996; Hall and Lane, 1994). On the other hand, because mutant p53

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protein has a longer half-life (~12 hr), and is present in relatively large amounts, it can be detected easily with or without antigen retrieval pretreatment (Figs. 10.7 and 10.8, respectively). In comparison with formalin fixation, alcohol fixation results in increased staining of p53 antigen, probably due to easy access of the antigen to the large antibody macromolecules in the absence of protein crosslinks. Allison and Best (1998) have compared the effects of alcohol fixation with those of formalin fixation, in conjunction with microwave heating, on the immunohistochemical demonstration of p53, PCNA, and Ki-67 antigens in oral squamous cell carcinoma. They indicate increased nonspecific staining of p53 antigen staining in the alcohol-fixed tissues. Similarly, fixed and treated tissues also showed p53 antigen staining in unexpected tissue components. Another adverse effect of alcohol fixation is the comparatively poor quality of cell morphology preservation, which becomes apparent at higher magnifications. Therefore, formalin fixation is preferred. Although a large number of immunohistochemical studies demonstrate that p53 overexpression is positively correlated with proliferation rates in many tumor types (Table 10.5), caution is warranted in the interpretation of such results because the presence of an heterogeneous population of cells within a tumor specimen is well known. This problem might be avoided by using cell lines derived from tumors, thus obtaining a homogeneous source of tumor cells. However, such cell lines might acquire mutations absent in the original tumor. Moreover, part of the positive immunoreactivity could result from an accumulation of wild-type p53 protein. Under certain circumstances, wild-type p53 protein may

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accumulate, probably because of complex formation with oncogenic proteins or as a response to DNA damage. Another problem is the presence of p53 staining in the cytoplasm in some cases, the significance of which is debatable (Linden et al., 1994; Bosari et al., 1993; Fisher et al., 1994). Immunohistochemical detection of wild-type and mutant p53 proteins can be carried out in fresh-frozen as well as formalin-fixed and paraffin-embedded tissues. This is best accomplished by using antigen retrieval with microwave heating or other types of heating such as autoclaving. A number of monoclonal antibodies are commercially available, the characteristics and sources of which are listed in Tables 10.3 and 10.4 and on pages 50–51. However, the validity of immunohistological analyses of p53 expression has been questioned. There are several factors, such as the source of antibodies and their crossreactivity, that are potentially responsible for inconsistent results. Demonstration of p53 in normal cells by immunohistochemistry (and flow cytochemistry) should be confirmed, when possible, by Western blot analysis (Nickels et al., 1997). As a general practice, an unusual staining pattern of p53 should be interpreted with caution.

USE OF MULTIPLE ANTIBODIES FOR LABELING P53 ANTIGEN
An antigen is a highly complex, three-dimensional molecule, and the precise location of epitopes on or within the antigen in most cases is not known. Furthermore, antigen retrieval methods may unfold the antigen molecule, exposing hidden (buried) epitopes.

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These methods may break down crosslinks between the antigen and the surrounding proteins, allowing the epitopes to become accessible due to the mechanism(s) responsible for the availability of epitopes for immunohistochemistry—a strategy to ensure that recognition of the epitope by the antibody is required. Such a protocol is presented below. A monoclonal antibody or its clones are directed against a specific amino acid sequence of epitope of the antigen molecule. If such an epitope is not accessible to a given monoclonal antibody, the interaction will not occur, resulting in false-negative immunostaining. To avoid this problem, multiple monoclonal antibodies against the same antigen but reactive to different amino acid sequences can be used. This can be accomplished by using more than one antibody simultaneously or separately. The approach almost ensures the interaction between at least one of the antibodies and its accessible epitope, resulting in positive immunostaining. It is also possible that more than one antibody in the cocktail of antibodies may interact with more than one epitope. As an example, three antibodies used in three separate studies for labeling p53 is described below. Seven monoclonal antibodies and one polyclonal antibody used against p53 antigen are given in Table 10.4. Each of the monoclonal antibodies shows specific affinity for a different range of amino acid sequences of the p53 molecule. By using three antibodies separately—DO-7 (for 21–25 amino acid sequence), Pab240 (for 213–217 amino acid sequence), and HR 231 (for 371–380 amino acid sequence)—false-negative immunostaining of p53 can be avoided (Tenaud et al., 1994). These three antibodies possess specificities for epitope distributed along the p53 molecule as shown in Figure 10.6.

Wild-Type p53 Antigen Retrieval Using Microwave Heating
The PAb 248 monoclonal antibody recognizes an epitope highly preserved between mouse and humans (Rotter et al., 1983), which thus can be used for localizing wild-type p53 antigen in human tissues. Using this antibody, wild-type p53 has been localized immunohistochemically in the normal human lymphoid and epithelial cells (Pezella et al., 1994). Sections of paraffin-embedded tissues are processed using standard antigen retrieval with microwave heating and the immunoperoxidase technique.

p53 Antigen Retrieval Using Microwave Heating
Tissues are fixed with 10% neutral phosphate–buffered formalin and embedded in paraffin, and sections ( thick) are mounted on poly-L-lysine-coated slides heated at 60°C for 30 min. The sections are deparaffinized in four changes of xylene and then rehydrated in a descending series of ethanol. Endogenous peroxidase activity is quenched by immersing the sections in 1% in distilled water for 5 min. The sections are rinsed in three changes of distilled water, transferred to a moist chamber, and covered with PBS. The slides are placed in 0.1 M sodium citrate buffer (adjusted to pH 6.0 with NaOH) in a plastic jar, which is transferred into a microwave oven. They are heated at 750 W for 17 min, with brief interruptions at 7 and 12 min to replace evaporated volume with distilled water. The slides are cooled to room temperature in the citrate buffer and transferred to PBS. Nonspecific background staining is blocked by treating the sections with diluted

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horse serum for 15 min at room temperature. After rinsing in PBS, the sections are incubated in DO-7 antibody (diluted 1:200 in PBS) in a moist chamber for 1 hr at room temperature. They are rinsed in PBS and treated successively with biotinylated horse antimouse antiserum and avidin-biotin peroxidase complex for 30 min each. This is followed by treating the sections with a solution of DAB (0.5 mg/ml) containing 0.009% hydrogen peroxide. The intensity of the brown reaction product is enhanced by immersing the sections in 0.125% osmium tetroxide. The sections are lightly counterstained with hematoxylin and successively immersed in acid alcohol, lithium carbonate solution, graded ethanol solutions, and xylene. The slides are coverslipped with Permount medium. Figure 10.7 shows the immunostaining of p53 antigen using microwave heating.

Frozen Section Immunohistochemistry of p53
Tissue specimens are snap-frozen at – 60°C in an isopentane freezing bath (Neslab, Portsmouth, NH) (Resnick et al., 1995). Sections thick) are cut on a cryostat,

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mounted on poly-L-lysine-coated slides, and immediately fixed with 100% ethanol at 4°C for 10 min, followed by air-drying for ~45 min. The slides are stored at –70°C for 12 hr, thawed to room temperature, and rehydrated in PBS (pH 7.4) for 5–10 min. Nonspecific reactivity is blocked by treating the sections for 15 min at room temperature with diluted horse serum ( Vector Mouse Elite ABC kit, Vector Labs, Burlingame, CA). This is followed by a blocking procedure for endogenous biotin (BioGenex avidin-biotin blocking kit, Vector Labs) according to manufacturer’s instructions. The sections are rinsed in PBS, followed by incubation in PAb 1801 antibody (1:100) for 1 hr in a moist chamber. They are rinsed in PBS and treated successively with biotinylated horse antimouse antiserum (30 min) and avidin-biotin-peroxidase complex (30 min) at room temperature, using the Vector Mouse Elite ABC kit. The chromogen, a mixture of DAB (0.5 mg/ml) and 0.009% is added. The intensity of the brown reaction product is enhanced by immersing the sections in 0.125% solution. The sections are lightly counterstained with hematoxylin and successively immersed in acid alcohol, lithium carbonate solution, graded ethanol solutions, and xylene. The slides are coverslipped with Permount medium. The results of this procedure are shown in Figure 10.9.

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Chapter 11

Estrogens

Endogenous estrogens are 18-carbon steroids and were discovered in the 1920s. They are produced in the ovaries, adrenals, and stroma of peripheral fat. is the dominant estrogen in reproductive-age women. Estrogens bind to members of the nuclear receptor superfamily. The functions of an estrogen are mediated by specific high-affinity estrogen receptors and located in the target cell nuclei. Estrogens classically exert their effects through the receptor mechanism of action. The estrogen enters the cells by passive diffusion and binds to the ER. Following a series of activation steps, the estrogen-ER complex, associated with the estrogen responsive element, functions as an enhancer for the estrogen-responsive, element-containing genes. Estrogen is a key intracellular modulator of the processes involved in differentiation, development, and homeostasis. This hormone produces physiological actions within a variety of target sites in the body and during development by activating a specific receptor protein. Estrogen plays a crucial role in embryonic and fetal development to influence female secondary sexual characteristics, reproductive cycle, fertility, and maintenance of pregnancy. In addition, estrogen modulates lipid and cholesterol homeostasis in females. The hormone also contributes to the neuroprotection seen in females after traumatic or ischemic cerebral insults (Roof and Hall, 2000). This neuroprotection can be partly explained by invoking estrogen’s lipid-lowering effect. Estrogen also directly affects the blood vessel wall, microvascular vasomotor tone, and production of vasoactive substances. Several mechanisms are responsible for these effects. Other putative effects of estrogens include preservation of autoregulatory function, an antioxidant effect, reduction of production and neurotoxicity, reduced excitotoxicity, increased expression of antiapoptotic factor bcl-2, and activation of mitogen-activated protein kinase pathways. Also, there is overwhelming data indicating that estrogens enhance survival of neurons both in vitro and in vivo (Green and Simpkins, 2000). Estrogens are synthesized not only in females but also in males. The synthesis of this hormone by cytochrome P450 aromatase in Leydig and Sertoli cells of the testis is well known (Carreau et al., 1999). This cytochrome is also found in the brain, where estrogen is important for imprinting male behavior (Beyer, 1999). There is clear evidence that the role of ER in males is associated with the maintenance of fluid reabsorption in the head of the epididymis (Hess et al., 1997). The loss of ER function in males interferes with the resorptive function of efferent ductules, a function that is essential for fertility (Hess, 2000).
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The biological importance of estrogen also becomes clear considering the number of disease states associated with altered production of this hormone or abnormalities in the manner in which the cell responds to the biological stimulus provided to the cell by estrogen. It is well known that estrogen replacement therapy is associated with numerous beneficial health effects, including a reduction in risk for cardiovascular disease, decreased incidence of osteoporosis, and a significant reduction in all-cause mortality (Grady et al., 1992; Hunt et al., 1990). Estrogen’s cardiovascular benefits result from improved profiles as well as favorable effects on the vascular wall. It has been suggested that nuclear factor-kB may be involved in both early and late stages of the inflammatory-proliferative process of atherogenesis, and the negative cross-talk between ER and this factor may be a fundamental mechanism in estrogen’s cardioprotection. Other mechanisms are discussed by Harnish et al. (2000). Estrogen use is also associated with a number of clinically relevant neurological benefits, including increased verbal memory, reduced incidence of Alzheimer’s disease, and decreased neuronal damage from stroke (Sherwin and Carlson, 1997; Paganini-Hill and Henderson, 1994; Schmidt et al., 1996). In addition, estrogen plays a positive role in inhibiting further progress of Parkinson’s disease (Saunders-Pullman et al., 1999). There are several possible explanations for estrogen’s effects on memory and cognition, including modulation of neurotransmitter function and increased synaptogenesis. The direct neuroprotective role of estrogens, as well as the proven clinical safety of these hormones, suggest that estrogen therapy may be useful in treating neurodegenerative diseases as well as neurotrauma such as head injury and cerebral ischemia, as mentioned above. While the role of estrogens and their receptors in breast cancer is discussed elsewhere in this chapter, it suffices to indicate that paradoxically, in addition to the initial promotor role of estrogens in breast cancer, they prevent spreading of cancer cells. The protective role of against cancer progression has also been presented elsewhere in this chapter.

ESTROGEN RECEPTORS
Three isoforms of the estrogen receptors (ER) have been identified, cloned, and characterized from several species: and (Green et al., 1986; Kuiper et al., 1996; Hawkins et al., 2000). These receptors are members of a superfamily of genes that consists of nuclear receptors for diverse hydrophobic ligands such as steroid hormones (estrogens, progestins, glucocorticoids, mineralocorticoids), retinoic acids (vitamin A), vitamin D, prostaglandins, and thyroid hormones. Most members of this family are ligand-dependent transactivators. After hormone binding and transformation, receptor-ligand complexes interact with specific hormone response element on target genes, regulating transcription. When not bound to the hormone, ERs exist in an unactivated, untransformed state (as a monomer) and complex with heat shock proteins. In the estrogen-binding state, the receptors undergo physico-chemical changes, including phosphorylation at specific serine and tyrosine residues that are accompanied by conformational changes (Arnold et al., 1997). These changes result in the dissociation of heat shock proteins from the activated complex and formation of a 5S homodimer with high affinity for estradiol and DNA. The transformed dimer binds to its specific estrogen response element located in the promoter region of estrogen-responsive genes, regulating their transcriptional activity. Estrogen

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receptor–estrogen response element interactions are augmented by the binding of coactivators or corepressors that further regulate gene transcriptional activity, without binding directly to DNA, via interaction with specific transcription factors such as Spl (Porter et al., 1997). Estrogen receptor was first identified in the 1960s, using binding assays that measured the uptake of radioactive estradiol by cytosolic homogenates of tissue (Jensen and Jacobson, 1962). The initial studies focused on rat tissues, but soon attention focused on the detection of ER in human breast cancers (Jensen et al., 1971). In the 1970s it became clear that the ER could be detected in 60–80% of human breast cancers and that it could be useful in predicting the response to endocrine therapy (McGuire, 1975). After 25 years this statement still holds true. Because biochemical methods used in the 1970s required large amounts of the tissue for homogenization, the studies concentrated on breast cancer (Fig. 11.1) rather than on normal breast (Fig. 11.2/Plate 5E). It was not until the development of antibodies against ER, which would be effective for the fixed tissue of a small size subjected to antigen retrieval, that normal breast tissue began to be analyzed for ER. Even today, a comparatively small number of studies are available on the ER in normal breast tissues. Consequently, we know much more about abnormal ER than about normal ER. Table 11.1 shows the presence of ER in a wide variety of carcinomas.

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Previously only a single type of ER was known to exist as the mediator of the genomic effects of estrogen in specific target tissues. Later, cloning of a gene encoding a second type of estrogen receptor was reported in the mouse, rat, and humans (Kuiper et al., 1996; Vladusic et al., 1998). To distinguish between these two ERs, the initial receptor is termed This development has prompted a reevaluation of the estrogen signaling system. In mammals, the gene is mapped to the q22-24 band of chromosome 14, while the gene is mapped to the long arm of chromosome 6 (Enmark and Gustafsson,

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1999). In addition, several isoforms of these two subtypes were recently reported. These are transcribed either by alternative exon splicing or usage of different promoters of a single gene (Friend et al., 1995; Chu and Fuller, 1997; Ogawa et al., 1998). Estrogen receptor gamma is derived through gene duplication. The existence of multiple forms of the receptor may explain the pleiotropic actions of estrogens in diverse tissues or species. The discussion in this volume pertains to receptors and and Estrogen receptor alpha and consist of a hypervariable N-terminal domain that contributes to the transactivation function (A/B), a highly conserved central domain responsible for specific DNA binding, dimerization, and nuclear localization (C), an estrogenbinding domain (E), a hinge region domain (D), and a domain (F) whose function is not known (Tonetti and Jordan, 1997). A high level of homology exists between and especially in the DNA-binding and estrogen-binding domains. These two ERs can form homodimers with themselves or heterodimers, providing three potential pathways for estrogen signaling. However, and differ in the C-terminal ligand–binding domain and in the N-terminal transactivation domain. The difference between ER subtypes in relative ligand binding affinity and tissue distribution explains the selective action of ER agonists and antagonists.

Estrogen Receptor Alpha
The human is a complex genomic unit exhibiting alternative splicing and promoter usage in a tissue-specific manner. This observation demonstrates the importance of transcriptional control in the regulation of expression. However, the mRNA stability is subject to hormonal control, suggesting that the regulation of the expression of this receptor may also occur at a posttranscriptional level (Saceda et al., 1989). Note that human mRNA has a relatively short half-life of approximately 5 hr in the breast carcinoma cell line MCF-7 after actinomycin D treatment; actinomycin D is the transcriptional inhibitor (Kenealy et al., 2000). Six functional regions (A–F) are recognized in the molecule, which show different degrees of amino acid sequence conservation. Human is comprised of 595 amino acids with a molecular weight of 66–70 kDa. Conserved domain organization responsible for specific functions of is DNA binding, ligand binding, dimerization, protein binding, and transcriptional activation. The hypervariable A/B domain in the amino-terminal region of exhibits little or no conservation between species. This region contains an activation function, is important for transactivation, and is responsible for gene and cell specificity. Region C corresponds to the DNA binding domain and is responsible for specific binding of the receptor to estrogen response elements located in target genes. Region D is the hinge region, which separates the DNA-binding domain from the ligand-binding domain. This region also facilitates conformational changes in the receptor molecule during activation and is important in receptor dimerization. Region D and the C-terminal portion of region C contain nuclear localization signals and are responsible for nuclear localization. Region E is located in the C-terminal portion of the receptor and is responsible for ligand binding. This region contains a second activation function domain, involved in transactivation in conjunction with A/B domain. The exact functional role of region E is not clear, although it may play

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a role in distinguishing between agonist and antagonist binding to the receptor molecule (Montano et al., 1995). is predominantly expressed in specific tissues, such as breast, uterus, and vagina. The receptor plays a key role in many normal physiological processes, ranging from female sexual development and reproduction to liver, fat, and bone cell metabolism. It is also involved in the biology of breast cancer and is used clinically as an important prognostic factor (Fig. 11.3). Significant amounts of the have been detected in more than 60% of human breast cancers. Approximately 70% of the tumors respond to antiestrogen therapy compared with only ~5% of the tumors. In spite of the usefulness of ER immunohistochemistry in the diagnosis and prognosis of the breast cancer, the published data are not always in agreement. The discordant immunohistochemical ER results reported in the literature are partly owing to the use of different monoclonal antibodies. Generally, different antibodies recognize a specific epitope within the domains over the entire length of the ER. For example, the difference between the reactivity of ERID5 monoclonal antibody (which targets an epitope in the A/B region) and H222 monoclonal antibody (which targets an epitope in the E region) observed in breast tumors is considered to be due to the presence of ER variants (Elias et al., 1995). Also, by developing monoclonal antibodies to specific domains of ER, the presence of structurally defective ER in breast tumor has been demonstrated (Traish et al., 1995). Thus,

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specific monoclonal antibodies can be used to identify dysfunctional ER in breast tumor biopsies. In fact, the failure of some ER-positive breast tumors to respond to endocrine therapy, the development of endocrine resistance, and progression in human breast cancer are related to the presence of ER variants. Caution is warranted in interpreting the status of breast cancer because a monoclonal antibody directed toward a certain ER domain may yield false-negative results if changes in the ER molecule have resulted in the absence or conformational alteration of the domain targeted. An improved approach to assess the ER status in the breast tumor is to use a panel of monoclonal antibodies that target several distinct epitopes within the ER domains. This approach has been successfully used for determining ER status in 46 primary breast carcinomas using three monoclonal antibodies (AER 311, ER1D5, and LH2) (Santeusanio et al., 2000). According to this study, in patients with breast tumors negative for these three antibodies, the disease progressed within 8 years from the diagnosis of the tumor, whereas all patients with tumors positive for all three monoclonal antibodies were alive 13 years after surgery. Considering the importance of ER variants in the immunohistochemistry of breast cancer, their characterization and functions are summarized below. mRNA undergoes alternate splicing, generating transcripts containing single, double, or multiple exon deletions. The presence of such transcripts in breast cancer cell lines and normal and malignant breast tissue specimens has been described (Leygue et al., 1996). Although the exact function(s) of these splice variants is not established, it is likely that these proteins differ in activity. Such proteins may differentially modulate the ER signaling pathway in normal tissues. Also, changes in the balance of these transcripts could perturb the ER signaling pathway and contribute to tumorigenesis, tumor progression, and response to hormone. Therefore, it is important to qualitatively investigate the difference in the levels and pattern of ER splice variant expression between normal and neoplastic tissues. Conventionally, the variants are characterized by coamplification with wild-type sequences using reverse transcription polymerase chain reaction (RT-PCR). However, this approach focuses on small regions of the known wild-type mRNA. Because of this threshold detection, spliced transcripts expressed at low levels may fall below the threshold of detection. To avoid this and other limitations of the conventional RT-PCR technique, the targeted amplification method can be used (Poola et al., 2000). This method involves the targeted amplification of the alternatively spliced molecules as separate gene populations using specific primers designed for the alternative splice junctions, without coamplification of wild-type molecules.

Estrogen Receptor Beta
Estrogen receptor beta has been cloned from rats, humans, and several other species (Kuiper et al., 1996; Mosselman et al., 1996; Lakaye et al., 1998). Human is expressed in multiple isoforms with various amino acid numbers. Recent studies document that the expressed full-length human is comprised of 530 amino acids (Fuqua et al., 1999). The DNA-binding domains of human and human are highly homologous, approaching 96%, while the ligand-binding domain shows only 59% homology. The N-terminal A/B domain, hinge region, and F domain are distinct in sequence between and The binds the natural hormone (estradiol) with affinity similar to

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that of However, it should be noted that the differences in the distribution and structure between these two receptors suggest that the two isoforms have different biological activity. The is present in the nucleus of a wide range of normal adult human and rat tissues, including breast, ovary, fallopian tube, uterus, lung, kidney, brain, heart, prostate, testis, oviduct, adrenal, seminal vesicle, and bladder (Saunders et al., 1997; Taylor and Al-Azzawi, 2000). The presence of and in the normal human breast tissue is shown in Figure 11.2. Specifically, this receptor is located in the epithelial cells in most male tissues, including the prostate, the urothelium and muscle layers of the bladder, and the Sertoli cells in the testis. In the uterus, both and are present in epithelial cells lining the lumen and glands. In the lung, is found in the cells lining the bronchioles and alveoli and smooth muscle.

Estrogen Receptor Gamma
It has been known for some time that some genomic actions of estrogen cannot be attributed to either or For example, continues to protect against vascular injury in both and knockout mice (Karas et al., 1999). This evidence suggests the presence of additional types of estrogen receptors. Recently, the presence of a third type of estrogen receptor, in a teleost fish, the Atlantic croaker (Micropogonias undulatus), was reported (Hawkins et al., 2000). This receptor is thought to have arisen through gene duplication from early in the teleost lineage. Receptors and are also present in this vertebrate species. The three ER subtype receptors are genetically distinct and have different distribution patterns in this vertebrate. These three subtypes of receptors have distinct functions, at least in the hypothalamus.

Distribution of Estrogen Receptors
is more widely distributed than and when both receptors are present in a tissue, the former is predominant. is present in the nucleus of a wide range of normal adult human and rat tissues, including breast, ovary, oviduct, fallopian tube, uterus, prostate, testis, seminal vesicle, bladder, and lung (Saunders et al., 1997; Taylor and Al-Azzawi, 2000). Based on mRNA analyses, this receptor is expressed in the central nervous system, cardiovascular system, immune system, and gastrointestinal tract (Gustafsson, 1999). In the lung, is found in cells lining the bronchioles and alveoli and smooth muscle. Moderate to high expression of is found in uterus, testis, pituitary, ovary, kidney, epididymis, and adrenal gland. In the uterus, and are expressed in epithelial cells lining the lumen and glands. Although distribution is closely related to the expression of in some tissues, the expression of these two receptors does not seem to be linked. Some cells lack while other cells show both of these receptors, and still other cell types are and A few examples follow. In the endometrium, both and are present in luminal epithelial cells and the nuclei of stroma cells, while expression is weak or absent in the endometrial glandular epithelia. In the

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ovary, is present abundantly in multiple cell types, such as granulosa cells in small, medium, and large follicles, theca and corpora lutea, whereas is undetectable in these cell types (Saunders et al., 1997). Breast and pituitary contain both receptors, whereas prostate shows positive immunoreactivity for but negative for An extensive list of the immunohistochemical distribution of and in adult human tissues is presented by Taylor and Al-Azzawi (2000).

ROLE OF ESTROGEN RECEPTORS IN BREAST CANCER
The ER and the progesterone receptor (PR) belong to the steroid hormone receptor family of ligand-inducible transcription factors, which play a key role in the development and progression of breast cancer. Although breast is influenced by many hormones and growth factors, estrogens play an important role in promoting the proliferation of both normal and neoplastic breast epithelium. The influence of estrogens on the proliferative activity of mammary epithelial cells is mediated by at least three mechanisms: receptor mediation, autocrine/paracrine loop, and negative feedback (Kumar et al., 1987; Huff et al., 1988; Soto and Sonnenschein, 1987). However, these mechanisms have not been precisely defined as to their role in the normal development and differentiation of the breast or in the initiation and progression of the neoplastic process. Because normal epithelium contains receptors for estrogen and progesterone, the receptor-mediated mechanism is a major player in the hormonal regulation of breast development. Considerable amounts of ER are present in more than 50% of primary human breast cancers. The presence of ER in primary tumors identifies patients with a lower risk of relapse and better overall likelihood of survival. Moreover, response to endocrine therapy mostly depends upon the presence of ER and PR, the latter indicating functional ER signal transduction because PR expression is regulated by ER. Consequently, ER determination has become an established procedure in the management of patients with breast cancer. Approximately 50–70% of patients with recurrent disease who had ER-positive primary tumors respond to hormonal treatment compared with only ~5% of patients with ER-negative tumors, suggesting a strong correlation between the growth of breast tumors in vivo and the presence of ER. However, the duration of response is limited because of progression to an estrogen-independent state of the tumor. In other words, patients with ER-positive breast cancer have a more favorable clinical course and prognosis and longer disease-free intervals than those with ER-negative cancer. Therefore, determination of the ER content of breast cancer tissue is indispensable for selecting a regimen of treatment when there is a relapse or for predicting the prognosis. Some prognostic factors predicting failure of endocrine therapy are known. The presence of epidermal growth factor (EGF) indicates poor prognosis and is correlated with lack of response to endocrine therapy in recurrent breast cancer. It is recognized that expression of EGF receptor is inversely related to ER expression in malignant breast tumors and breast cancer cell lines, both at the protein and mRNA levels (Klijn et al., 1992). However, ~50% of ER-positive tumors contain EGF receptors. But ER and EGF receptors are rarely expressed simultaneously in the same malignant cell. The likely reason is that both receptor signal pathways become uncoupled during malignant progression. This and other evidence documents the heterogeneous nature of primary breast tumors.

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A double immunohistochemical method has been used for determining the expression patterns of ER, PR, and EGF receptors in breast biopsies (Van Agthoven et al., 1994). It was demonstrated that ER/PR and EGF receptors in breast tumor cells were inversely related at the single cell level. However, the expression of these three receptors in individual normal luminal cells was not mutually exclusive.

Breast Cancer and Tamoxifen
It is well known that the ER level is important as a prognostic and predictive marker in breast cancer patients. ER status is correlated with endocrine therapy. Tamoxifen is the most common partial antiestrogen and is used for treating all stages of breast cancer. Clinical trials demonstrate that tamoxifen is useful for the treatment of breast cancer (Fisher et al., 1998). It acts by competitively binding to ER, but its activity ranges from full estrogen antagonist to a partial agonist in different tissues. Its effectiveness varies with the prevailing estrogenic environment (Furr and Jordan, 1984). The effectiveness of tamoxifen can be evaluated in relation to Ki-67 antigen (a nuclear proliferation marker), which is useful in determining the prognosis of breast cancer. (Ki-67 antigen is discussed in Chapter 9.) The relationship between ER levels and Ki-67 antigen expression before and after tamoxifen treatment has been investigated (Dardes et al., 2000). Immunohistochemical studies demonstrate a decreased Ki-67 labeling index after tamoxifen treatment in ER-positive patients. Patients with down-regulation of ER expression also show decreased Ki-67 labeling index after tamoxifen therapy. This phenomenon may be based on the ability of tamoxifen to induce apoptosis and reduce the levels of ER as a transcription factor (Dardes et al., 2000) This and other studies indicate that short-term (4 weeks) tamoxifen therapy decreases the proliferation of breast cancer in ER-positive breast tumor specimens. In relation to ER level there is no difference in the Ki-67 labeling index level between pre- and posttamoxifen treatment of ER-negative patients. Recent studies suggest that postmenopausal patients older than 50 with ER-negative breast cancer, who do not respond well to either hormonal therapy with tamoxifen or adjuvant chemotherapy, may have a significant response to vaccination with autologous tumorassociated antigens (Jiang et al., 1999). Such a vaccination results in a reduction in serum IL-6 concentration in patients with ER-negative breast cancers; it is known that estrogen represses IL-6 expression. This approach does not have a direct cytotoxic effect on cancer cells but is an attempt to promote mechanisms of rejection of the tumor by the host.

ANTIBODIES
Estrogen receptor comprises several structural domains with specific and overlapping functions. A number of monoclonal and polyclonal antibodies are available, some of which show affinity for specific domains of the ER molecule. The antibodies discussed below are efficient tools for ER immunohistochemistry on sections of formalin-fixed, paraffinembedded tissues and facilitate the cellular site expression of and receptors in human and rat tissues. Most of these antibodies are used for labeling ERs in breast tissue in conjunction with pretreatment with antigen retrieval methods.

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Monoclonal antibody ERID5 (AMAC Laboratories, Westbrook, ME) is produced by using recombinant human ER (instead of purified ER) for immunization (Al Saati et al., 1993). It detects but not The antibody reacts with the A/B region of the aminoterminal domain of This antibody also reacts with several truncated forms that are translated from slice variant mRNA, such as those involving Del. 5 and/or Del. 7. Its affinity also extends to the 67-kDa polypeptide chain of estrogen obtained by transformation of Escherichia coli and transfection with COS cells with plasmid vectors expressing estrogen. In comparison with antibody H222, antibody ERID5 is more sensitive, produces higher nuclear staining intensity, and can be used at a higher dilution (1:100) (Fig. 11.4). The higher sensitivity of ERID5 translates into a better correlation with the biological behavior

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of breast cancer than does H222. According to Goulding et al. (1995), however, using the Spearman’s rank correlation method, a highly significant correlation is found between the H scores using antibody ERID5 or antibody H222. For manual immunohistochemistry with antibody H222, the sections are stained using Abbott’s ER-ICA monoclonal antibody kit. H222 was the first antibody applied to paraffin sections. Another monoclonal antibody, NCL-ER-6F11, has been generated by using a recombinant ER protein instead of the peptide antigen approach (Bevitt et al., 1997). Because multiple epitopes are presented by the recombinant protein at immunization, production of a greater number of hybridomas is expected. NCL-ER-6F11 antibody is specific to human (see Fig. 11.3) and does not bind to recombinant human NCL-ER-6F11 antibody compares favorably with ERID5 antibody, which is also generated using recombinant ER. Mouse monoclonal antibody AER311 (Neomarkers, Fremont, CA), unlike ERID5, recognizes the carboxyl-terminal domain and reacts only with wild-type estrogen, except for DNA-binding truncated protein (Huang et al., 1996). Immunohistochemistry using ERID5 or AER311 can distinguish hormone-binding truncated protein from wild-type estrogen. The estrogen-enzyme immunoassay method recognizes various mutant proteins that can also be detected only by the ERID5 antibody and not by the AER311 antibody because these two antibodies recognize different targets on the ER molecule. As stated above, the AER311 antibody does not react with ERs that lack the hormone-binding domain. Immunohistochemical studies using these two antibodies have shown that a number of palpable breast cancers lack the carboxyl terminal in the ER, regardless of wild-type ER mRNA expression (Hori et al., 1999). Other monoclonal antibodies include D75P3, CC4-5 (Ventana Medical Systems) and 6F11 (Vector Laboratories, Burlingame, CA). Compared with CC4-5, 6F11 gives more intense nuclear staining and less cytoplasmic reactivity. Saunders et al. (1997) have raised a polyclonal antiserum using a peptide specific for The peptide (CLSKAKRNGGHAPRVLEL) corresponding to amino acids 196-213 of rat was conjugated to keyhole limpet hemocyanin and used to immunize rabbits according to standard procedures. Polyclonal IgGs were purified from serum on a Hitrap protein A Sepharose column based on the manufacturer’s instruction (Pharmacia). The monoclonal mouse antibovine (05-394) antibodies directed against SDSsolubilized calf uterus and polyclonal rabbit antirat (06–629) antibodies developed against the N-terminal region of the human sequence are commercially available (Upstate Biotechnology, Lake Placid, NY). Another polyclonal rabbit antirat
(310) antiserum developed against the C-terminal region of the human is also commercially available (Affinity Bioreagents Inc., Golden, CO). sequence

To test possible interactions between various ER functional domains, monoclonal antibodies to various regions of the ER have been developed (Traish and Pavao, 1996). Monoclonal antibody F9 was developed against a synthetic 30-mer hybrid oligopeptide. Another monoclonal antibody, NMT-1, was raised against 15-mer peptide from the Nterminal A/B region (amino acids 240–154). Monoclonal antibody 213 was generated against peptide AT3 in the DNA-binding domain (amino acids 247–263). The effects of binding these site-directed, monoclonal antibodies to specific regions of the ER molecule on the conformation of this molecule have been determined. Such studies indicate that the conformational change within a small stretch of the ER molecule

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caused by the binding of an antibody is transmitted to another distal region of the receptor. This phenomenon is exemplified by the binding of the antibody NMT-1 to the A/B region of the receptor, which causes the release of the antibody from its epitope in the DNA-binding region. Thus, the binding of these site-directed, monoclonal antibodies to specific regions of the ER molecule affects the conformation of this molecule.

IMMUNOHISTOCHEMISTRY
Validation of the immunohistochemical method for detecting ERs is through clinical correlation studies. The ultimate usefulness of this methodology depends on its ability to predict clinical outcome, especially the response to hormone therapy. Based on the evaluation of the clinical relevance of measuring ER status with immunohistochemistry, there is a statistically significant relationship with clinical outcome. In addition, by comparing the ER status measured in the same tumor, using both immunohistochemistry and biochemical ligand-binding assays, 80–90% agreement has been found between these two tests (Clark, 1996). Positive results with respect to ERs obtained in breast cancer tissue with immunohistochemistry is a sign of good prognosis related to higher survival durations. Estrogen and progesterone receptor levels are frequently negative in malignant tumors with metastases. Positivity thresholds of ERs for both immunohistochemistry and the DCC assay vary among different published studies. This threshold for immunohistochemistry ranges from 5–30% positive nuclei in breast cancer among laboratories. In women, the amount of 10 fmol/mg of protein as the cutoff point for defining ER positivity for biochemical ligandbinding assays is accepted worldwide. This amount establishes the relationship between the amount of cytosol protein and the probability of hormone dependence of the tumor. Although the application of immunohistochemistry to benign tumors and malignant tumors is highly specific, in some cases it may be less sensitive (Martin de les Mulas et al., 2000). A reliable method of measuring the ER content in human breast cancer is important for optimal treatment and a qualified estimate of the recurrence-free survival of the patient. The majority of the studies on the expression of ERs, especially in human tissues, have been accomplished using RNA techniques such as reverse transcription–polymerase chain reaction (RT–PCR) and in situ hybridization. Although the RT-PCR method is an effective tool to describe the presence of a particular gene in the tissue, this approach does not indicate the specific cell that expresses the gene. In situ hybridization overcomes the problem of cellular localization, but it is difficult to relate the expression of a particular mRNA to the expression of the functional protein. Moreover, this method is difficult to carry out. Immunohistochemistry, on the other hand, is a relatively simple technique that overcomes these problems by identifying the precise cellular localization of the functional protein. This technique, using paraffin sections, provides information on the ER status of tumors very simply and rapidly. In addition, this approach is superior to frozen section immunohistochemistry, the dextran-coated charcoal assay (DCC) (see page 276), or the enzyme-linked immunosorbent assay (ELISA) for predicting the response to endocrine therapy. It has been demonstrated that a significant enhancement of the predictive power for response to tamoxifen on relapse is achieved by immunohistochemical estimation of both

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receptors, ER and PR (PgR) (Barnes and Millis, 1995). Note that although ER or PR nuclear expression alone predicts response to hormonal therapy, combined ER/PR phenotypes have more precise predictive ability than either factor alone. Prognostic and predictive factors in breast cancer commonly assessed by immunohistochemistry are reviewed by Mohsin and Allred (1999). Recently, Taylor and Al-Azzawi (2000) have carried out an extensive immunohistochemical investigation identifying human tissues that express receptor and comparing the expression pattern of with that of The detection of immunohistochemical staining semiquantitatively is preferred over other methods because by using specific, monoclonal antibodies the ER positive cells are identified by a distinct nuclear labeling. The staining can range from weak to intense, depending upon the number of recognizable ERs present in the individual nuclei. In fact, some evidence indicates that computerized quantitation is no better than semiquantitative visual analysis (Schultz et al., 1992; Remmele and Schicketanz, 1993). Also a strong correlation has been found between results obtained with true-color, computer-assisted image analysis and semiquantitative scoring (Kohlberger et al., 1999). However, other studies indicate that computerized image analysis is superior to semiquantitative assessment because of higher accuracy and reproducibility (McClelland et al., 1991). Quantitative immunostaining analysis of ER using the Cell Image Analysis System SAMBA 4000 has been carried out (Esteban et al., 1994 a, b, c; see also page 105). This system optimizes measurements, while human deficiencies are reduced to a minimum. Moreover, semiquantitative scoring requires more experience, know-how, and training. Also, ER immunohistochemistry unfortunately has not been subjected to rigorous statistical analysis to define cutoff values associated with clinically meaningful endpoints such as response to endocrine therapy. Automated electronic analysis in the near future will establish reliable observer-independent evaluation of immunohistochemical variables. Although some of the computer-assisted image analysis equipment is too expensive for daily, routine use, it is possible to analyze ER and PR expression routinely and inexpensively with good correlation to clinical outcomes using a relatively inexpensive standard IBM PC and Adobe Photoshop software (Lehr et al., 1997). A similar type of analysis has been carried out on endometrial samples from patients treated with hormone replacement therapy to help predict clinical outcomes (Wahab et al., 1999). A number of variations of the immunohistochemical methodology are available to localize ERs. The expression of these receptors can be studied on paraffin sections or frozen sections, with or without antigen retrieval application, although the latter application is preferred. Various heating treatments, such as microwave heating, autoclave heating, and boiling on a hot plate, are equally effective in unmasking ERs in paraffin sections. An example of the distribution of ERs in the pituitary gland is given below. Using immunohistochemistry in conjunction with autoclave antigen retrieval, cellular distribution of and has been accomplished on paraffin sections of fetal and adult rat pituitary glands (Nishihara et al., 2000). The expression of in the fetal pituitary is lower than that during the adult period and is limited to the nuclei of anterior lobe cells from day 17 of gestation. In contrast, is present in the nucleus as well as in the cytoplasm in both the anterior and posterior lobes during the fetal period from day 12 of gestation. The distribution of in the adult pituitary is mainly restricted to the anterior lobe. It seems that plays different roles in the pituitary during the fetal and adult periods. The above evidence also indicates that oncogenetic changes in the expression of these

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receptors are not uncommon. Note that the quantitation of prognostic markers, including ERs, is hampered by a time-related loss of antigenicity, especially in paraffin sections on glass slides (see page 84).

COMPARISON OF IMMUNOHISTOCHEMISTRY WITH BIOCHEMICAL LIGAND-BINDING ASSAYS
Biochemical ligand-binding assays (e.g., dextran-coated charcoal [DCC] assay) were technically and clinically validated more than a decade ago. In fact, most of our knowledge about the ER in breast cancer prior to the 1990s was obtained by using these assays. However, these tests are more limited than immunohistochemistry; the advantages of immunohistochemistry include its being easier to perform, less expensive, safer, and faster. Also, immunohistochemistry is applicable to a wide variety of specimens (e.g., cytological preparations, frozen tissue blocks, formalin-fixed archival tissue blocks, and paraffinembedded tissue blocks and sections). This method, in addition, provides direct correlation with cell morphology. Another advantage of immunohistochemistry is that tissues of a small size (e.g., biopsies) can be used. This is important because it is better to detect tumors at an early stage, when they are small. The necessity of early detection cannot be overemphasized. Very small tumors and fine-needle aspirates cannot be used for biochemical assays. Although the DCC assay provides quantitative results, it does not take into account the relative amount of the connective tissue in the specimen, the presence of carcinoma in situ lesions, or normal ducts and lobules. These limitations are not encountered when using paraffin sections. In addition, immunohistochemistry allows the use of archival tissues when fresh tissues are not available. This method does not require any special, expensive equipment and can be carried out in any standard laboratory. An additional important advantage of immunohistochemical detection of ERs is the precise histological identification of tissue structures, both tumoral and nontumoral. Falsepositive results obtained with the DCC method resulting from the presence of remnants of normal glandular structures or small dysplastic areas surrounding the tumor can be identified using immunohistochemistry. Also, the DCC assay can yield false-negative results because the amount of tissue containing the ERs may be too small in the specimen to be detected. In contrast, immunohistochemistry is able to identify even a tiny tissue containing the ERs. Note that immunohistochemistry measures a fundamentally different property of the ER than that obtained with the DCC assay; the former detects the presence of an antigenic epitope, whereas the latter indicates the ability to bind a specific hormone. It is possible that the hormone binding site may be damaged or nonfunctional, but the immunoreactive epitope of the ER molecule may still be preserved. The immunohistochemistry of ERs has been exhaustively compared with the DCC assay. Review of the literature indicates ~85% agreement between these two methods (Allred, 1999). This is true when immunohistochemistry is restricted to fresh-frozen sections. Immunohistochemistry of frozen sections compared with paraffin sections is a more specific test to detect ER-positive tumors with very low tumor cellularity; the DCC assay gives false-negative results for such tumors. A number of publications have also reported good agreement between the DCC assay and immunohistochemistry of paraffin-embedded

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breast cancer tumors and cytological preparations (Allred et al., 1990; Masood, 1989; Shimada et al., 1985). However, immunohistochemistry of paraffin sections is both less sensitive and reproducible compared with frozen-section immunostaining. One of the disadvantages of frozen sections is poor preservation of cell morphology. For this and other reasons, the use of paraffin sections has become more popular than frozen sections. Note that controversies over the technical and clinical validation of immunohistochemistry have not been completely resolved. Whether or not this method should completely replace biochemical ligand-binding assays remains controversial. Despite this cautionary statement, it is true that the specificity of immunohistochemistry is theoretically valid because it is based on the use of well-characterized monoclonal antibodies raised against epitopes restricted to the ERs.

Dextran-Coated Charcoal Assay
The dextran-coated charcoal (DCC) assay measures the hormone-binding capacity of the cytosol fraction of the tumor tissue (Lee and Chan, 1994). Fresh tissue (more than 100 mg) is immediately frozen, homogenized, and the cytosol fraction is extracted. Aliquots of the cytosol are incubated with radiolabeled estradiol with and without 100-fold molar excess of nonradiolabeled competitor (diethylstilbestrol) to displace radiolabeled steroid from the low-capacity specific binding sites (ER), but not from the high-capacity nonspecific binding sites in the cytosol. The unbound steroid is removed by selective adsorption on DCC. The receptor binding capacity, reported in femtomoles of radiolabeled steroid bound per milligram of cytosol protein, is calculated by subtracting the level of nonspecific cytosolbound radioligand (radiolabeled steroid with excess of competitor) from the total cytosol-bound radioligand (radiolabeled steroid without competitor).

SEMIQUANTITATIVE ASSESSMENT OF ESTROGEN RECEPTORS
Two methods are available to determine the estrogen (and progesterone) status in the tissue samples: biochemical assays (e.g., dextran-coated charcoal [DCC] method) and immunohistochemistry. Immunohistochemistry is very useful in estimating prognosis and monitoring therapy in breast cancer. An advantage of immunohistochemistry over the DCC assay is that the former can be used with small tissue samples, and it also facilitates morphological evaluation of individual tumor cells. In contrast, because ligand-binding assays only detect estrogen receptors in an unoccupied form, they are prone to interference by other endogenous hormones and may give rise to unreliable results. Furthermore, such assays preclude assessment of estrogen receptor contents of individual cells. Scoring can be performed according to the two different semiquantitative methods of Remmele and Stegner (1986) and Reiner et al. (1987). Specific staining is identified by distinct colored staining of nuclei with the histoscore system, which considers both intensity of staining and percentage of stained cells. According to the method of Remmele and Stegner (1986), intensity is graded from 0–3, where 0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining. The percentage of stained cells is categorized as follows: 0 = 0%, 1 = 1–10% ,

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2 = 11–33%, 3 = 34–55%, and 4 = 67-100%. The two values obtained from the staining intensities and percentage of positive cells are multiplied. This method results in values of 0 (negative) and 1, 2, 3, 4, 6, 8, 9, or 12 (positive). On the other hand, according to the second system (Reiner et al., 1987), the score is acquired by adding the two values (staining intensities and percentage of positive cells) and obtaining the values 0 (negative), 2 and 3 (low positive), 4 and 5 (intermediate positive), or 6 and 7 (high positive). In contrast to the complex Remmele-score, the Reiner score is relatively simple and easily applicable, making it more advantageous in daily routine investigations (Biesterfeld et al., 1996). Moreover, the Reiner score has better reproducibility than the Remmele score. It is emphasized that at least three different fields on a slide are observed, and 100 cells are counted in each field. More than one observer should participate independently in the scoring in a blind test. Following scoring, a consensus score should be established among the observers. Note that immunohistochemical reactions do not always yield homogeneous results but are subject to variations from slide to slide and from case to case. Because tumors are heterogeneous, caution is warranted in assuming that a particular section of the tumor is representative of the whole tumor (see page 14 for discussion on tumor heterogeneity).

IMMUNOSTAINING OF ESTROGEN RECEPTORS IN PROSTATE TISSUE
Sections ( thick) of formalin-fixed and paraffin-embedded tissues are deparaffinized with xylene and rehydrated with ethanol (Bonkhoff et al., 1999). Endogenous peroxidase is blocked by treating the sections with 0.3% hydrogen peroxide. The sections are placed in 10 mM citrate buffer (pH 6.0) and heated in a microwave oven at 750 W for 5 min followed by at 450 W for 5 min. They are washed in PBS and treated for 30 min with normal rabbit serum. After being washed in PBS, the sections are incubated overnight in mouse monoclonal antibody NCL-ER-6F11, diluted 1:200 (Novocastra) in a humid chamber. This antibody is obtained by recombinant protein preparation from MCF-7 cells and is directed against the full-length molecule. Following rinsing in PBS, the sections are incubated for 30 min in the secondary biotinylated rabbit antimouse immunoglobulin (Dako). This is followed by applying the horseradish peroxidase–labeled avidin-biotin complex (ABC-HRP) method according to the manufacturer’s instructions (Dako). To amplify the signal and enhance immunodetection of the estrogen, the biotinylated tyramine method is applied. After precipitation of the biotinylated tyramine for 10 min through the enzymatic action of horseradish peroxidase and hydrogen peroxide (0.1%), the biotin precipitate is detected with an additional application of the horseradish-labeled avidin biotin complex (Dako) for 30 min in a humid chamber. The peroxidase reaction is developed with DAB (Sigma) to form a brown endproduct. Negative controls are performed on consecutive sections by replacing the primary antibody with a nonimmune mouse serum. Figure 11.5 (Plate 5F) shows staining of using NCL-ER-6F11 antibody. To localize on paraffin sections, 65-kDa antirat ER antibodies are used (Upstate Biotechnology, Lake Placid, NY). This antibody is obtained by immunizing rabbits with synthetic peptides representing the N-terminal amino acid residues 46–63 of human The deparaffinized sections on slides are placed in the Target Retrieval Solution (pH 6.1)

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(Dako) and heated in a microwave oven as described above. After incubation in normal swine serum (Dako) for 30 min, the sections are incubated in the 65-kDa antirat estrogen antibodies, diluted 1:100 with PBS for 12hr. Detection is achieved as described above, except that the secondary biotinylated rabbit antimouse immunoglobulin is replaced with the biotinylated swine antirabbit antibody (Dako). To localize in frozen sections, antigen retrieval with heating is not required. 210-180-C050 antibodies (Alexis Corporation, Nottingham, UK) are obtained by immunizing rabbits with synthetic peptides representing the C-terminal amino acid residues 467–485 of human estrogen.

IMMUNOSTAINING OF ESTROGEN AND PROGESTERONE RECEPTORS IN FINE-NEEDLE ASPIRATES OF BREAST
The ThinPrep smear with microwave antigen retrieval pretreatment is a reliable method for estrogen (and progesterone) analysis in breast carcinoma (Leung and Bédard, 1999). The smears are prepared from fine-needle aspirates of patients with breast carcinoma according to the directions in the Operator’s Manual (Cytyc, Boxborough, MA). The smears are postfixed for 5 min with Surgipath cytology fixative (Surgical Medical Industries, Richmond, IL) and kept frozen at —70°C until the antigen retrieval step. Silanized-coated slides are used for better adhesion of cells. Target Retrieval Solution (Dako S1700) is diluted 1:10 with distilled water and adjusted to pH 6.0. ThinPrep smears are rehydrated in 95% ethanol and distilled water for 5 min each. The smears are placed in a plastic slide container with sufficient retrieval solution. The container is immersed in a sufficient quantity of warm water inside the microwave pressure cooker (Dako, DO300X) and heated in a microwave oven for 20 min at full power until steam is generated. The smears, along with retrieval solution, are allowed to cool for 10 min. The smears are removed and rinsed with warm water, followed

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by Tris-saline buffer (TSB). Immunostaining is performed using the Dako K1900 ER/PR staining kit (Dako Corporation, Carpinteria, CA). It includes all ready-to-use reagents, antibodies, and detection system. The smears are covered with hydrogen peroxide for 5 min, then with distilled water, and then placed in TSB for 5 min. They are incubated in primary monoclonal antibodies (Dako K1900) for 30 min and then washed with TSB for 5 min. This is followed by incubation with biotinylated linking antibody for 10 min and washing in TSB for 5 min. The smears are incubated with strepavidin-peroxidase for 10 min and then washed with TSB for 5 min. They are treated with chromogen substrate DAB for 10 min and rinsed with tap water for 5 min. Counterstaining is accomplished with hematoxylin for 20 sec. The smears are dehydrated, cleared and mounted. The results of this procedure are shown in Figure 11.6.

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Chapter 12

Her-2 (c-erbB-2) Oncoprotein

The HER-2/neu oncogene plays a key role in breast and many other cancers. Methods, including immunohistochemistry, are available to analyze tumor HER-2 status (Fig. 12.1). Elucidation of the human genome is expected to significantly improve many aspects of health care, especially the diagnosis and management of cancer. Genetic discoveries are already resulting in the development of specific, effective, and less toxic cancer drugs than many of those in current use. An important example of a less toxic reagent is Herceptin, which targets HER-2/neu gene product, p185 (HER-2).

HER-2/NEU ONCOGENE
HER-2/neu gene was first discovered as a transforming oncogene in a series of ethyl nitrosourea–induced rat neuroblastomas, where it was called neu (Schecter et al., 1984). Approximately 15 years ago, this oncogene was isolated independently by two separate groups, which named it HER-2 (Coussens et al., 1985) and c-erbB-2 (Semba et al., 1985), respectively. Further evidence revealed that the two genes were the same (Schechter et al., 1985), and it was renamed HER-2/neu. The gene is located on chromosome 17 at q21 and encodes a 185-kD glycoprotein composed of cytoplasmic, transmembrane, and extracellular domains. This tyrosine kinase glycoprotein has 40% sequence homology to the epidermal growth factor receptor (EGFR) but is distinct from the EGFR. A physiological ligand for this presumed receptor remains unidentified. In other words, the kinase may function as a cellular receptor for an undiscovered ligand; however, it has been suggested that 17-estradiol may mimic ligand activity for this oncogene protein (Matsuda et al., 1993). Amplification and overexpression of the HER-2/neu gene has been reported in a wide variety of tumor types, predominantly of epithelial origin, including those of female and male breasts (Slamon et al., 1989; Fox et al., 1991), ovary (Slamon et al., 1989; Hellström et al., 2001), colon (D’Emilia et al., 1989), pancreas (Thybusch-Bernhardt et al., 2001), stomach (Jain et al., 1991), salivary gland (Kernohan et al., 1991), bladder (transitional cell carcinoma) (Wood et al., 1991), head and neck (Riviere et al., 1991), and prostate (Schwaab et al., 2001). This gene has also been reported in malignant cartilage (Wrba et al., 1988), papillary thyroid carcinoma, and uterine cervical carcinoma (Hale et al., 1992). HER-2/neu has also been implicated in pulmonary carcinomas (Hirashima et al., 2001),
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colorectal adenocarcinomas (Koeppen et al., 2001), skin cancer (Krähn et al., 2001), and lung cancer (Cox et al., 2001). The amplification of the HER-2/neu oncogene is thought to have significance in the early stages of malignant transformation, especially in the breast and ovary, and thus may be considered a prognostic marker of these carcinomas. A statistically significant correlation between the amplification of this gene and survival of patients with breast and ovarian tumors has been reported (Slamon et al., 1989; Carr et al., 2000). Because the gene is overexpressed in breast cancer patients, determination of its prognostic significance is being evaluated. This oncogene is indeed an independent prognostic indicator of a subset of breast cancers that are at high risk of early recurrence, regardless of tumor grade, estrogen/progesterone receptor status, or lymph node status (Carr et al., 2000). However, according to Slamon et al. (1987), association of HER-2/neu amplification and poor prognosis is stronger for patients with lymph node metastases than without lymph node involvement. The HER-2/neu gene is overexpressed in 25–30% of human breast cancers, and in ~90–95% of these cases, the overexpression is a direct result of gene amplification. The amplification or overexpression of this gene is associated with shorter disease-free survival as well as overall survival. Appropriate follow-up studies have confirmed the prognostic association between the HER-2/neu gene alteration and clinical outcome for both nodenegative and node-positive breast cancers (Press et al., 1997) The HER-2/neu gene is also overexpressed in epithelial ovarian cancer. Several studies have reported that overexpression of this gene is associated with poor survival rates in

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advanced epithelial ovarian cancer (Berchuck et al., 1990; Bast et al., 1992). Anreder et al. (1999) have also reported poor survival rates for patients showing overexpression of the oncogene in the ovarian surface epithelial tumors. Note, however, that some types of primary tumors, such as breast cancer, may contain elevated p185 protein or HER-2 mRNA levels in the absence of detectable gene amplification, suggesting that in some cases gene amplification is not strictly required for p185 expression. In other words, the presence of this protein in some cancer cells itself is an index of tumor aggressiveness, regardless of HER-2/neu amplification (Dati et al, 1990). It should be noted that although HER-2/neu gene is considered to be the target gene for amplification at chromosome bands 17ql2–q21, the amplicon harbors several closely located genes such as retinoic acid MLNs 50, 51, 62, and 64, gastrin, hormone and topoisomerase (Järvinen et al., 1999). Many breast tumors with HER-2/neu amplification show simultaneous amplification or deletion of topoisomerase gene. Amplification of the HER-2/neu is followed by complex secondary genetic aberrations, which lead to amplification or deletion of the topoisomerase gene in a majority of tumors. A simple molecular mechanism suggested for this phenomenon is that the amplification of the chromosomal segment includes both genes. The importance of this information is that the gene copy number aberrations of topoisomerase may divide HER-2-amplified breast tumors into clinically meaningful entities.

HER-2 ONCOPROTEIN
Her-2/neu is a protooncogene encoding a 185 kDa protein (HER-2). HER-2 is one of the epidermal growth factor receptor (EGFR) family of four closely related transmembrane growth factor tyrosine kinase receptors. These are designated HER-1 to HER-4 (c-erbB-1 to c-erbB-4), and they exhibit a high degree of homology to each other. Transmembrane HER molecules exist as inactive monomers on the cell surface but form receptor dimers that are stabilized by ligand binding to their extracellular domain domain). Dimerization can occur between the same receptor (a homodimer) (Alroy and Yarden, 1997). The resulting phosphorylation of tyrosine residues initiates complex signaling pathways that ultimately lead to cell division. The interaction between the HER monomers and various ligands (e.g., epidermal growth factor and transforming growth factor), and the ensuing diversity of signal transduction from the intracellular tyrosine kinase domain, results in a complexity that explains the key role played by this type I growth factor receptor family in regulating cell growth and differentiation (van de Vrjver, 2001). It is possible that the function of HER-2 is to stimulate growth after the formation of heterodimers with other members of the HER family. In fact, a class of ligands, neuregulins, bind to HER-3 and HER-4, causing heterodimerization with HER-2 (Carraway et al., 1994). HER-2 is the signaling subunit and has no independent ligand. Antibody blockade of HER-2 prevents heterodimerization, eliminating neuregulinstimulated signaling. The inhibition of this signal may induce tumor regression and delay return to a tumor growth phase in patients with aggressive breast cancer. It is known that HER-2 signaling enhances metastasis in breast cancer cells by inducing endothelial cell retraction, a process that appears to precede endothelial transmigration (Carter et al., 2001). In other words, breakdown of the vascular barrier caused by HER-2

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signaling may be sufficient to facilitate tumor cell transmigration and metastasis. The molecular mechanism(s) of HER-2-induced endothelial cell retraction is unknown. It is interesting to note that expression and secretion of an aberrant HER-2 splice variant has been reported in various cell lines and tissues, which can interfere with the oncogenic HER-2 activity (Aigner et al., 2001). Expression of this truncated 100-kDa HER-2 variant encodes the extracellular domain of HER-2 and inhibits growth factor–mediated tumor cell proliferation. The exact role played by this variant during the progression of human cancer is not clear.

HER-2 OVEREXPRESSION
Overexpression of the HER-2 protein is one of the most studied molecular changes that occur in human cancer. The main cause of HER-2 overexpression is HER-2/neu gene amplification, although protein overexpression has been observed in some subsets of breast carcinoma despite a lack of gene amplification (Persons et al., 1997). It is reasonable to suggest that HER-2 may not have an identical role in all tumors. It is known that HER-2 overexpression is able to transform cells in vitro, although catalytic activation of this receptor may also be involved in other functions. In fact, the precise functional significance of this phenotype is uncertain. This uncertainty is reinforced by evidence that HER-2 overexpression shows stronger association with preinvasive rather than with advanced disease. Moreover, extensive apoptosis occurs in preinvasive diseases. This and other evidence raises doubts about the precise significance of the HER-2 phenotype in vivo. Published immunohistochemical studies of overexpression of HER-2 report a wide range of overexpression rates, ranging from 9 to 60%. The likely reason for this variation is the use of different antibodies, tissue types, and fixation and staining protocols. In addition, different scoring systems result in interpretation differences. To minimize these variations the use of a uniform immunohistochemical method, such as the HercepTest, is recommended. The HercepTest kit provides standardized procedure and evaluation criteria.

SIMULTANEOUS OVEREXPRESSION OF HER-2 AND P53
Among molecular markers for sporadic breast cancer, p53 protein and HER-2 receptor protein have retained special attention. Genetic alterations resulting in overexpression of these two proteins are a frequent finding in early stages of invasive ductal carcinomas of the breast, suggesting a role in breast cancer pathogenesis and possibly a function in disease progression. The detrimental consequence of simultaneous expression of these two proteins has been found even in node-negative mammary carcinomas (Albanell et al., 1996). Moreover, HER-2 and p53 overexpression individually correlates with a higher growth rate in mammary carcinomas. More recently, Rudolph et al. (2001) also provided evidence that overexpression of either HER-2 or p53 resulted in an increased cycling ratio, which is thought to be additive in tumors overexpressing both proteins. p53 expression is significantly more prevalent in young patients, whereas HER-2 expression shows only a trend toward a higher occurrence in the young.

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DISTRIBUTION OF HER-2 IN CARCINOMAS
In addition to breast cancer, HER-2/neu gene amplification is common in ovarian, prostate, and gastric cancers, while a relatively low incidence of amplification is found in a large number of cancers arising in other organs, which are summarized below. The association of HER-2 and the prognosis of breast cancer patients have been studied most extensively. The amplification of this gene and overexpression of HER-2 oncoprotein have been found to be associated with poor clinical outcome. However, whether HER-2 is an independent prognostic factor is controversial.

Astrocytic Tumors
It is difficult to distinguish between benign and malignant astrocytic tumors. Some lowgrade astrocytomas behave similarly to anaplastic ones. This problem may be solved through determining the proliferative index of antigens such as HER-2, p53, PCNA, p21, EGF, and EGFR. Such an immunohistochemical study has been recently carried out by Bian et al. (2000). This study indicates that aberrations of HER-2, p21, EGF, and EGFR might be early events in the initiation and progression of astrocytomas, but they are unrelated to histological grade and prognosis of astrocytomas. On the other hand, p53 overexpression is involved in all the stages, while PCNA might be important in evaluating astrocytoma malignancy. In this study, sections were pretreated with 0.1% trypsin containing 0.1% prior to treatment with 0.3% in methanol to block endogenous peroxidase.

Bladder Carcinoma
Wood et al. (1991) have used the Southern hybridization method for detecting DNA amplification and a possible structural rearrangement of the HER-2/neu oncogene in 1 of 12 bladder tumors. Amplification of this oncogene in the tumor was sixfold that of oncogene found in placental DNA. Approximately 36% of the tumors studied overexpressed HER-2 mRNA, which was 3- to 38-fold that of normal urothelium. HER-2 overexpression occurred in superficial and invasive tumors. Deoxyribonucleic acid amplification occurs infrequently in bladder carcinoma, in contrast to its occurrence in some other carcinomas. Immunohistochemical analysis has shown that p185 HER-2 polyclonal antibody is specific for HER-2 protein overexpression in bladder carcinoma. This study was carried out prior to the use of Herceptin.

Ewing's Sarcoma
Ewing’s sarcoma is the second most common malignant bone tumor in children. The majority of patients have microscopic metastases at diagnosis; the lung is the most common metastatic site. This sarcoma is a relatively rare disease with limited therapeutic options. The majority of patients are initially responsive to chemotherapy with vincristine, doxorubicin (Adriamycin), and cyclophosphamide. However, relapsed disease is usually extremely difficult to treat because of its resistance to chemotherapy (Zhou et al., 2001).

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Clinical studies indicate that overexpression of HER-2 correlates with poor prognosis and shorter patient survival in osteosarcoma (Onda et al., 1995). There is a close association between HER-2 overexpression and resistance to chemotherapeutic agents (Tsai et al., 1995). Zhou et al. (2001) have investigated HER-2 expression in three different human Ewing’s sarcoma cell lines (TC71, RD, and A4573). It was found that this protein is overexpressed in the three cell lines. This study also indicates that E1A gene therapy may provide a new approach for treatment of this carcinoma. It has been demonstrated that transduction of TC71 cells with the ElA gene using an adenoviral vector downregulates HER-2 overexpression in these cell lines (Zhou et al., 2001). Downregulation of HER-2 increases apoptosis in tumor cells that overexpress the oncogene.

Intrahepatic Cholangiocellular Carcinoma
Immunohistochemistry has been used for investigating the expression of HER-2-4 in 38 intrahepatic cholangiocellular carcinomas (Ito et al., 2001). HER-2 expression was observed in more than 50% of the cases but was not related to any clinicopathological features. HER-3 expression was linked to lymph node metastasis, and HER-4 expression was directly related to proliferating activity and lymph node metastasis.

Laryngeal Squamous Cell Carcinoma
Preembedding immunoelectron microscopy has been used for localizing HER-2 at the single cell level in laryngeal squamous cell carcinoma (Grzanka et al., 2000). HER-2 was located both on the plasma membrane and in the cytoplasm of cancer cells in approximately half of the cases investigated (a total of 15 surgical specimens). The polyclonal antibody, rabbit antihuman c-erbB-2 (Dako), was used in this study, which recognizes HER-2 protein on the membrane as well as in the cytoplasm. Membrane and cytoplasmic staining of HER-2 has also been reported in gastric carcinoma (Lee et al., 1994). According to Grzanka et al. (2000), a significant correlation between HER-2 in laryngeal squamous cell carcinoma and pathological characteristics, such as nodal status and histological grade, was not found.

Non-Small-Cell Lung Carcinoma
In addition to carcinomas in other organs, HER-2 oncoprotein is overexpressed in cancers of the lung. Lung cancer is currently the leading cause of cancer-related death. Most patients have inoperable tumors at the time of diagnosis, and metastatic relapse in patients with operable non-small-cell lung carcinoma remains a frequent event. Genetic abnormalities in lung cancer frequently include the mutation, rearrangement, or overexpression of several genes and their protein products, such as HER-2, p53, and K-ras. The HER-2 protein is strongly stained immunohistochemically on the cell membrane of adenocarcinoma, and the overexpression of this protein relative to that of normal alveolar lung tissue is associated with a worse prognosis. A soluble form of the HER-2 protein is also found in serum.

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It is interesting to note that some HLA-class 1 antigens confer resistance to the progression of bronchogenic carcinoma. There is also a relationship between survival time and HLA for epidermoid lung carcinoma (Prazak et al., 1990). Also, an association between HLA-DR7 and resistance to lung cancer has been reported (Romano et al., 1991). Analysis of this relationship is important for determining not only the etiology but also the appropriate therapy and prognosis of lung cancer. Recently, Yoshimura et al. (2000) carried out a detailed study of this relationship in lung cancer. This study suggests that HER-2 is correlated with prognostic factors for lung cancer independent of HLA-associated genetic factors. Immunohistochemical studies demonstrate overexpression of HER-2 protein in non-small-cell lung carcinoma (NSCLC). The protein is located predominantly on the plasma membrane, although its location has been reported in the cytoplasm, especially in adenocarcinoma. In a recent immunohistochemical and fluorescence in situ hybridization study, with the exception of occasional single tumor cells, cytoplasmic HER-2 expression was not seen (Cox et al., 2001), in contrast to some other studies employing different antibodies and other processing parameters (Tateishi et al., 1991). In lung cancer, overexpression of HER-2 is associated with a poor prognosis. Unlike breast carcinoma, HER-2/neu gene amplification in NSCLC is uncommon. However, in advanced lung carcinoma, the Herceptin is positive. Thus, Herceptin may have a role in the treatment of a proportion of patients with this disease, but it is of a limited clinical value, especially in the adjuvant setting.

Ovarian Carcinoma
Approximately 25% of primary ovarian carcinoma expresses HER-2 protein, but unlike its expression in breast cancer, it is controversial to what extent HER-2 amplification and overexpression correlate with prognosis. However, HER-2 expression is more frequent in ovarian carcinomas relapsing after chemotherapy (Meden et al., 1998). Recently, it was shown that tumor lines established in vitro from ovarian carcinomas, as well as ovarian carcinoma cells harvested from malignant ascites, frequently overexpress the HER-2 protein at their surface (Hellström et al., 2001). This evidence suggests that cells expressing this protein have a selective growth advantage over HER-2-negative cells. To further clarify the above-mentioned suggestion, ovarian SKOV3.A2 cells have been used for identifying the specific HER-2 chain for a better source of its epitopes (Castillega et al., 2001). It is known that these cells express high and stable levels of HER-2. In these cells, HER-2 is not autophosphorylated at Y1248 and other receptor tyrosine kinase (RTK) sites. As a consequence, the proliferation of these cells is less dependent on mitogenic signaling by tyrosine-phosphorylated HER-2 and is consequently less susceptible to inhibition by geldanamycin. This drug is known to mediate its inhibitory effects on RTK by binding strongly to stress proteins such as heat shock protein or glucose-regulated protein, which forms complexes with HER-2. Such an inhibition occurs indirectly by destabilizing stress protein–complexed kinases (Chavany et al., 1996). Geldanamycin does not inhibit HER-2 mRNA and protein synthesis in tumor cells, but by dissociating the HER-2-glucose–regulated protein complex, it reduces the protein half-life. Treatment of cells with this drug increases the rate of ubiquitination of existing HER-2 protein with consequent

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faster proteasomal degradation. Also, HER-2 synthesized in the presence of geldanamycin is unstable and does not accumulate in tumor cells (Mimnaugh et al., 1996). Like patients with breast carcinoma, those with ovarian cancer may benefit from treatment with Herceptin in combination with chemotheropeutic drugs. Herceptin treatment is effective, in both early and advanced stages of ovarian cancer (when the majority of tumor cells express HER-2 protein), for eliminating the potentially more malignant HER-2-positive tumor cells. The effectiveness of Herceptin is based on the affinity of this monoclonal antibody for the extracellular domain of HER-2, which is common as ovarian carcinomas progress. However, a tumor vaccine inducing antibody and/or T-cell immunity to HER-2 epitopes will ultimately provide the most effective means to prevent the emergence of HER-2-positive cells.

Prostate Carcinoma
Prostate carcinoma is the most common cancer in men in the United States. Approximately 180,400 new cases were diagnosed in 2000, with 31,900 deaths as a result of the disease. Prostate cancers typically begin with androgen-dependent lesions, but the cancer usually becomes androgen-independent when the disease is advanced. It has been shown, for example, that androgen-independent LAPC-4 prostate cancer sublines express a higher level of HER-2 than that expressed by their androgen-dependent counterparts (Carter et al., 2001). Alterations in the signal transduction pathways mediated by HER-2 play an important role in the progression of prostate cancer. This protein is normally expressed at a very low level in a few human secretory epithelial cells but is overexpressed in a number of human cancers, including prostate carcinoma. Overexpression of HER-2 is an indicator of poor prognosis in prostate cancer patients. Because of the deaths and considerable clinical complications that occur as a result of prostate cancer, metastatic forms of the disease have been and remain an important target for novel therapeutic interventions. Although standard hormonal therapy for metastatic prostate cancer has a high response rate (70–80%), hormone resistance ultimately develops, which necessitates additional therapy. Novel therapeutic HER-2-specific immunotoxins, such as scFv (FRPS)-ETA, have been developed and might be useful agents for the treatment of prostate cancers with high levels of HER-2 protein. Another example is the development of bispecific antibodies (e.g., MDXH210) designed to direct the cytotoxic effects of monocytes and macrophages to destroy tumor cells expressing HER-2. These constructs are discussed elsewhere in this chapter.

Squamous Cell Carcinoma of Cervix
An inverse pattern of expression of HER-2 and epidermal growth factor receptor (EGFR) is found in normal tissues of the female genital tract and placenta (Wang et al., 1992). Although HER-2 is closely related in structure to EGFR, these two oncoproteins may play a different role in cell control. Ngan et al. (2001) have studied the expression of HER-2 and EGFR at the protein level using immunohistochemistry, at the RNA level

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applying the ribonuclease protection assay, and at the DNA level using the Southern blot and hybridization method. Activation of proto-oncogenes can be determined by assessing the level of RNA expression or DNA amplification. A large number of patients with squamous cell carcinoma of the cervix were recruited. EGFR showed a high percentage (74.2%) of overexpression with immunohistochemical staining and 35.4% of DNA amplification in squamous cell carcinoma of the cervix. In contrast, HER-2 showed only 19 8% of overexpression with staining and 17.2% of DNA amplification. It is concluded that the abnormal expression of these two proteins has no prognostic significance on survival rates.

METHODS FOR DETECTING HER-2 STATUS
A number of methods are available for analyzing tumor HER-2 status. The selection of the method depends on the target molecule to be detected. The target molecules are DNA mRNA, and protein (Fig. 12.2). HER-2 gene amplification can be detected by Southern blot (Press et al., 1994), slot blot (Naber et al., 1990), and dot blot assays (Descotes et al., 1993), fluorescence in situ hybridization (FISH) (Persons et al., 1997), in situ hybridization (ISH) on isolated nuclei or tissue sections (Smith et al., 1994), and polymerase chain reaction (Gramlich et al., 1994). Assays to determine mRNA overexpression include Northern blot (Slamon et al., 1989), Western blot (Press et al., 1994), slot blot (Naber et al., 1990) and ISH (Naber et al., 1990). Methods to assess HER-2/neu protein product overexpression

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include Western blot analysis (Slamon et al., 1989), immunoassays (Dittadi et al., 1997), FISH (Lebeau et al., 2001), and immunohistochemistry (Pauletti et al., 2000). Some of the above-mentioned methods have limitations. Southern blot analysis requires a large amount of high-quality DNA, and the results are unreliable if the sample has a low percentage of tumor cells (Ross and Fletcher, 1998). For these and other drawbacks, this method is precluded from becoming a routine clinical protocol to determine HER-2 status. Polymerase chain reaction (PCR), on the other hand, has several advantages: it can be used to analyze small numbers of tumor cells, DNA from formalin-fixed, paraffinembedded tumor tissue can be used, and it can be automated and standardized. Quantitative PCR techniques are currently being assessed for their clinical application to HER-2 DNA testing (Vona et al., 1999). However, presently the PCR technology is not optimally suited for routine, clinical application (see next section for details of quantitative analysis of HER-2/neu expression). It is apparent that many of the above-mentioned methods to analyze gene amplification and mRNA overexpression are beyond the scope of most pathology laboratories for technical reasons, and most of these assays require prospective collection of fresh tissue and thus are not applicable to archival tissue specimens. Fluorescence in situ hybridization is more widely used than Southern blot and PCR techniques and allows visualization of HER-2 DNA in individual cells using a specific fluorescence-labeled probe. Recently, Pauletti et al. (2000) have demonstrated HER-2/neu gene amplification in infiltrating breast adenocarcinoma using FISH with a HER-2/neuspecific probe. Another elegant study has demonstrated three-color FISH images of breast carcinoma cell nuclei from two primary tumors with HER-2 oncogene amplification (Järvinen and Liu, 2000). By performing dual-color FISH, the HER-2/neu gene status can be quantified, and information about the ploidy status of chromosome 17 can also be obtained (Walch et al., 2001). The detection of gene amplification in individual tumor cells is one of its major advantages. However, in the absence of standardization, results will differ. For details about detecting HER-2/neu amplification with FISH, see Ross et al. (2001). This detection method in breast cancer is based on the Oncor INFORM HER-2/neu Gene System using a sequence biotinylated probe (Oncor, Gaithersburg, MD).

QUANTITATIVE ANALYSIS OF HER-2/NEU GENE EXPRESSION
It is well established that formalin-fixed, paraffin-embedded tissue is the most widely available specimen for retrospective clinical studies. These specimens provide an invaluable source for the elucidation of disease mechanisms and validation of differentially expressed genes as therapeutic targets or prognostic indicators. However, the reliable quantitation of gene expression in formalin-fixed, paraffin-embedded tissues has serious limitations. Nucleic acids may be extracted from these specimens. Although this is a lesser problem for DNA, RNA isolated from such tissues is of poor quality due to its degradation before fixation is completed. Furthermore, formalin fixation crosslinks nucleic acid and proteins and covalently modifies RNA by adding monomethylol groups to the bases. This modification becomes a problem in subsequent RNA extraction, reverse transcription, and quantitative analysis.

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To cope with the above-mentioned problem, quantitative gene expression analysis can be accomplished in combination with laser-assisted microdissection in formalin-fixed, paraffin-embedded tissues. Using an optimized RNA microscale extraction procedure in conjunction with real-time quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) based on fluorogenic TaqMan methodology, Specht et al. (2001) have analyzed the expression of a panel of cancer-relevant genes, including HER-2/neu. They used 54 microdissected nonneoplastic and neoplastic archival samples from patients with Barrett’s esophageal adenocarcinoma for analyzing HER-2/neu expression with the QRT-PCR and compared it with that obtained in parallel with fluorescence in situ hybridization and immunohistochemistry. The results of these three methods matched one another. The QRT-PCR has been used for comparing quantitative mRNA expression of HER-2/neu with DNA and oncoprotein levels in the metaplasia-dysplasia-adenomacarcinoma sequence of Barrett’s adenocarcinoma (Walch et al., 2001). Tissue sections from the same area can be used for QRT-PCR of laser-microdissected tumor cells and for immunohistochemistry. The method has been successful in quantifying HER-2/neu gene expression in small microdissected tissue samples from archival material as well as in premalignant lesions (Specht et al., 2001; Walch et al., 2001). Only a locus-specific HER-2/neu gene amplification is associated with strong mRNA overexpression and strong plasma membranous immunostaining in Barrett’s adenocarcinoma. Quantitative PCR techniques are currently being assessed for their clinical application to HER-2 DNA testing (Vona et al., 1999). However, at present PCR technology is not optimally suited for routine, clinical application. The QRT-PCR is a sensitive, accurate, and highly reproducible method for studying gene expression. The method is based on the 5' nuclease activity of Taq DNA polymerase and involves cleavage of a specific fluorogenic hybridization probe that is flanked by PCR primers spanning an amplicon range of 60–150bp (Gibson et al., 1996). It is capable of detecting PCR products as they accumulate during amplification. The reactions are characterized by the point during cycling when PCR amplification is still in the exponential phase, allowing precise quantitation of RNA over a wide dynamic range. Because of the small target size, this approach is suitable for quantitative determination of gene transcript levels, even in tissue extracts containing partially fragmented RNA. Because only small amounts of RNA are required, this technique is applicable to small clinical biopsies and microdissected cell clusters from frozen or formalin-fixed, paraffin-embedded tissue sections (Specht et al., 2001). Laser-assisted microdissection is indispensable for the selective analysis of stroma-free tumor cell populations, circumventing the problem of tissue heterogeneity as well as providing the possibility of assigning characteristic gene expression patterns to particular histological phenotypes (Simone et al., 1998). This methodology opens new avenues for the investigation and clinical validation of gene expression changes in archival tissue specimens.

DETECTION OF HER-2 ONCOPROTEIN
Three main techniques have been used for detecting HER-2 oncoprotein overexpression: Western blot analysis, enzyme-linked immunosorption assay (ELISA), and immunohistochemistry. Western blot analysis is limited to basic research rather than routine clinical

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analysis of HER-2 status because it requires fresh tumor homogenates. The use of tumor homogenates results in large variations, depending on the amount of stromal and other nontumor cells present in the homogenate, causing a potential dilution effect (Ross and Fletcher, 1998). Enzyme-linked immunosorption assay can be used to measure HER-2 protein in tissue homogenates or in serum. It is a relatively simple technique and is well suited to automation (van de Vijver, 2001). However, when tumor cytosolic fractions are used, histological information is lost and invites the undesirable dilution effect. Therefore, the ELISA assay is not used routinely to determine HER-2 status. On the other hand, immunohistochemistry facilitates the identification of HER-2 protein overexpression in individual tumor cells and has become the most common approach for determining HER-2 status. Numerous reasons for its advantages are detailed throughout this book. As is true in many other techniques, routine immunohistochemistry has the disadvantage of being nonstandardized. One way to significantly minimize interlaboratory variability and achieve a degree of standardization is to use a single commercial test kit such as the HercepTest (Dako) discussed below. Another limitation of immunohistochemistry is that it relies on subjective interpretation of staining results. Many variables affect staining results, which are discussed in Chapter 5 and elsewhere in this book. Table 12.1 indicates one variable, i.e., antibodies, that affects HER-2 protein positivity. This table also indicates the role of different antibodies in determining HER-2 positivity in breast tumors, using immunohistochemistry. In conclusion, immunohistochemistry and FISH are recommended for HER-2/neu analysis in both routine clinical practice and in clinical research studies, using sections of formalin-fixed, paraffin-embedded tissues. Although these two methods show a high level of correlation with each other in the evaluation of HER-2/neu status of breast cancer, immunohistochemistry is preferred for routine use. The FISH procedure requires more time and expense than immunohistochemistry. In addition, the FISH slides must be stored at –20°C or lower and are prone to quenching of the fluorescent signal with time. More important, immunohistochemistry provides information on immunostaining as well as on cell morphology of the same section. However, according to Pauletti et al. (2000), FISH provides superior prognostic information in segregating high-risk from low-risk breast cancers than that obtained with immunohistochemistry. Antibodies used in this procedure

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are discussed later. Excellent studies comparing these two methods have been carried out by Jacobs et al. (1999) and Lebeau et al. (2001). Controversy continues over whether HER-2/neu gene amplification or gene product p185 overexpression is the best predictor of therapy response and clinical outcome. Because of this uncertainty, it is apparent that further clinicopathological studies are required to demonstrate whether HER-2/neu amplification or protein overexpression, or a combination of the two methods, has prognostic value in breast cancer. The most widely applied techniques to detect the amplification of this gene and the overexpression of its protein product are FISH and immunohistochemistry, respectively. The ideal approach is to correlate quantitative FISH with immunohistochemistry using antigen retrieval and computer-assisted image analysis. The results are further improved when immunohistochemical studies are assessed as the difference between tumor cells and nonneoplastic breast tissue. In other words, immunostaining intensity of nonneoplastic breast tissue is subtracted from the neoplastic staining, correcting for cytoplasmic background staining. Only distinct membranous immunostaining of HER-2 has prognostic value in breast cancer, while occasional cytoplasmic staining is without such relevance. Both techniques allow the study of small amounts of formalin-fixed, paraffin-embedded tissue and the interpretation of the findings on a cell-by-cell basis. Using this approach, Lehr et al. (2001) have convincingly demonstrated a high degree of concordance between the two techniques in breast cancer.

BISPECIFIC ANTIBODIES
Bispecific antibodies are chemically or genetically linked antibodies with two heterologous antigenic specificities. Potentially antineoplastic bispecific antibodies can be prepared by combining specificities for a tumor antigen and cytotoxic trigger molecules on immunoeffector cells. Such studies were developed in order to target cytotoxic immunoeffector cells to tumors to facilitate antibody-dependent cell toxicity (see also Chapter 2). Essentially, bispecific antibodies (e.g., MDX-H210) are novel antibody constructs designed to direct the cytotoxic effects of monocytes and macrophages to the destruction of tumor cells expressing HER-2. Recently, Sen et al. (2001) have described methods for generating highly effective HER-2-specific cytotoxic T cells by arming activated T cells with anti-CD3 X anti-HER-2 bispecific antibody. In this method OKT3 and 9184 anti-HER-2 monoclonal antibodies were conjugated and used to arm T cells that were subsequently tested for binding, cytotoxicity, and cytokine secretion assays. Armed T cells aggregate and selectively kill HER-2-positive breast cancer cells (MCF-7). Such cytotoxic T cells can be produced by arming activated T cells with nanogram quantities of OKT3/9184. Advantages of this technique are that arming activated T cells with low doses of the bispecific antibody obviates the need for administering large amounts of the antibody required for infusional therapy, and cytotoxicity is augmented by combining antibody targeting and T cell–mediated killing. Also, binding of effector cells at the tumor site by armed activated T cells may augment tumoricidal activity, as well as increase local cytokine secretion leading to recruitment of other immune effectors. This approach is unique; in future clinical trials, billions of activated T cells could be armed with milligrams of the bispecific antibody, thus becoming HER-2-specific cytotoxic T lymphocytes.

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A recombinant fusion protein [scFv(FRP5)-ETA] consisting of an HER-2-specific single-chain antibody and the Pseudomonas exotoxin A has been developed for evaluating its cytotoxicity on HER-2 expressing an established human prostate cancer cell line (LNCaP) (Wang et al., 2001). This cell line expresses high levels of HER-2 protein. Exposure of these cells to scFv(FRP5)-ETA causes significant cell death and reduces the level of prostate-specific antigen. Based on this evidence, scFv(FRPs)-ETA might be considered a useful agent for treating human prostate cancer cells with high levels of HER-2 expression.

Bispecific Antibody MDX-H210
MDX-H210 is a partially humanized Fab' X Fab' bispecific antibody constructed by chemical conjugation of the F(ab') fragments of the murine monoclonal antibody 520C9 (anti-HER-2) having specificity for the cell surface region of the HER-2/neu gene product and H22, a humanized monoclonal antibody that binds to the human immunoglobulin receptor (CD64). is a 72-kDa protein that is one of three receptors expressed on the plasma membrane of monocytes, macrophages, and or G-CSF stimulated PMNs (Lewis et al., 2001). Monclonal antibody H22 binds to a site outside the ligand binding domain for IgG yet effectively triggers cellular responses in the presence of high concentrations of human (serum) IgG. In other words, cytotoxicity is not blocked by IgG or serum. Lewis et al. (2001) have studied the combination of with MDX-H210 based on the hypothesis that would activate and up-regulate the expression of on neutrophils, monocytes, and macrophages. This approach facilitates an interaction between expressing effector cells and tumor cells expressing HER-2, thus enhancing destruction of tumor cells. Humanized MDX-H210 has dual specificity and immunological activity in vitro; that is, it causes lysis of HER-2-expressing cell lines mediated by monocytes and and G-CSF-activated PMNs. Recently, a phase 1 pilot trial of the MDX-H210 in patients whose prostate cancer overexpressed HER-2 was carried out by Schwaab et al. (2001). They undertook this trial to direct the cytotoxic effects of MDX-H210 on monocytes and macrophages through to destroy prostate tumor cells expressing HER-2. This agent was well tolerated at doses that appeared to be immunologically and clinically active. At these doses biological activity was demonstrated and characterized by binding of the antibody to circulating monocytes, release of monocyte-derived cytokines, a decrease in circulating HER-2 protein, and short-term stabilization of prostate-specific antigen levels. However, patients receiving an intravenous infusion of MDX-H210 did show malaise, fever, chills, and myalgias. A larger phase 2 study of this antibody combined with cytokine (GM-CSF) is underway by these workers. In another recent phase 1 trial, MDX-H210 was combined with for increasing the expression of as well as activating the monocyte/macrophage immunoeffector cells in patients with advanced cancer that overexpressed HER-2 (Lewis et al., 2001). Among the 23 patients, 19 had breast cancer, 3 had prostate cancer, and 1 had lung cancer. This study indicated the absence of a positive relationship between maximum plasma concentration of MDX-H210 or peak plasma cytokine concentrations and clinical toxicity.

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VACCINES
Recently, there has been renewed interest in developing vaccines for use in cancer treatment. The main factors giving impetus to this therapeutic approach include a better understanding of the immune system, the identification of several T cell–specific tumor antigens, more effective adjuvants, and the ability to construct more immunogenic molecules using recombinant DNA techniques (Murray et al., 2000). Current vaccine strategies for the treatment of solid tumors tend to focus on the cellular arm of the immune response. The overexpression of HER-2 protein in cancer cells makes it an ideal target for vaccines and other targeting strategies. Vaccines optimized to induce maximum T cell immunity to HER-2 may lead to potent in vivo antitumor immunity. HER-2 protein has been evaluated as a potential target for the development of cancer vaccines because preexistent T cell and antibody responses to HER-2 have been described in breast cancer patients (Disis and Cheever, 1996). In other words, breast cancer patients have preexisting immunity to the HER-2 receptor in the form of elevated antibody titers and T cell immunity. Elevated anti-HER-2 T cell responses have been demonstrated in breast and ovarian cancer patients following immunization with peptides derived from the HER-2 protein (Disis et al., 1999). However, whether peptide-specific T cell responses can be translated to antitumor immunity has yet to be established. A recent study has utilized an in vivo murine tumor model expressing human HER-2 for evaluating potential HER-2 vaccines consisting of either full-length or variable subunits of HER-2 delivered in either protein or plasmid DNA form (discussed later) (Foy et al., 2001). The mechanism of protection elicited by plasmid DNA vaccination appears to be exclusively CD4-dependent and not CD8- or antibody-dependent, whereas the protection observed with intracellular domain protein vaccination requires both CD4 and CD8 T cells. However, the exact mechanism(s) responsible for immunity to DNA has not been elucidated. Another recent study supports the use of the dendritic cell-based vaccine as a therapeutic strategy to target both CD4 and CD8 T cells to HER-2 (Chen et al., 2001). To minimize the possibility of deleterious effects, the transforming activity of the HER-2 molecule can be inactivated by a single amino acid substitution (lysine to alanine), unlike other studies in which the entire intracellular domain was removed.

Genetic Immunization
Genetic immunization, also known as DNA or polynucleotide immunization, is a novel strategy for vaccine development in the host. In this technique plasmid DNA encoding either individual or a collection of antigens is directly administered to a host. As a result, the delivered foreign gene is expressed by the host, which in turn leads to the induction of a specific immune response against the in vivo produced antigen. DNA immunization has been shown to induce protective immune responses in several infectious disease and cancer experimental model systems (Cohen et al., 1998). These vaccines have also entered the clinical trials both as protective and as therapeutic agents. Unlike peptide vaccines, which must be specific for each individual’s MHC, expression of plasmid-encoded tumor antigens within the host antigen-presenting cells following vaccination results in the presentation of multiple tumor–associated epitopes in the context

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of MHC class I and/or class II molecules. Direct intramuscular injection with DNA plasmid expressing HER-2/neu tends to induce antigen-specific cellular and humoral responses in mice (Wei et al., 1999). Chen et al. (1998) have shown that plasmid DNA encoding a truncated rat erbB-2/neu that lacked the intracellular domain could induce protective immunity against erbB-2/ neu–expressing mammary tumors as effectively as plasmid encoding the full-length erbB-2/neu oncogene. However, more recent clinical trials demonstrated that CD4 T cells recognizing peptides from both extracellular and intracellular domains of human erbB-2/neu protein can be induced by peptide vaccination. Vaccination with full-length DNA through ex vivo targeting of the dendritic cells has the advantage of presenting the complete repertoire of erbB-2/neu epitopes in association with MHC class I and class II molecules, maximizing the number of peptide epitopes available for T cell recognition (Chen et al., 2001). DNA vaccination requires host dendritic cells for priming the T cell response, either through direct transfection or antigen transfer from transfected nonhematopoietic cells. Because dendritic cells are functionally impaired in cancer patients, antigen processing and presentation following genetic immunization may be inefficient. However, this problem can be overcome by differentiating ex vivo dendritic cells from precursors present in the peripheral blood. The ex vivo cultured dendritic cells are fully functional and can be used as cellular vectors for vaccines (Dhodapkar et al., 1999).

IMMUNOHISTOCHEMISTRY
Immunohistochemistry can be used, with or without antigen retrieval, for localizing HER-2/neu gene protein (p185). In the absence of antigen retrieval, immunoelectron microscopy can also be employed for detecting this protein in different cell types at the ultrastructural level. Recently, this method was used for detecting p185 in laryngeal squamous cell carcinoma with the electron microscope (Grzanka et al., 2000). The use of rabbit antihuman HER-2/neu oncoprotein (Dako) (diluted 1:100) demonstrated antigenicity not only on the plasma membrane, but also in the rough endoplasmic reticulum. It is known that the protein moiety of this glycoprotein is synthesized in the rough endoplasmic reticulum, and glycosylation subsequently occurs in the Golgi complex. Most studies have been carried out using immunohistochemistry with the light microscope. HER-2/neu gene protein (p185) expression can be investigated with the mouse monoclonal antibody (mAbl) (Triton Diagnostic, Alameda, CA), an IgGl immunoglobulin that recognizes the external domain of this protein. This antibody has been used at a dilution of 1:200 for targeting this gene product in primary breast cancer tissue, without antigen retrieval pretreatment (Fig. 12.3) (Horiguchi et al., 1994). Alternatively, HER-2/neu gene product expression can be localized in breast cancer tissue using the rabbit polyclonal antiserum (R60), which targets the intracellular domain of p185 (Pauletti et al., 2000). Tissue sections do not require an antigen retrieval treatment. Another polyclonal antibody, Anti-c-erb-B2, can be used in conjunction with antigen retrieval in a microwave oven according to the standard procedure presented in this volume. This protocol has been used for targeting c-erbB2 (HER-2/neu) gene protein in the ovarian surface epithelial tumors (Anreder et al., 1999). Polyclonal antibody pAbl (Triton Biosciences) can also be used for assessing immunostaining (both the intensity and

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extent) of the (HER-2/neu) gene product in breast cancer tissue (Kay et al., 1994). For detecting this gene product in bladder, lung, and renal tumors, monoclonal antibody NCLCB11 has been used (Kay et al., 1994). This antibody recognizes the internal domain (cytoplasmic staining) of the HER-2/neu gene product. The expression of this oncoprotein has also been studied in mammary Paget’s disease using three different antibodies (Edorh et al., 1995). Antibodies NCL-CB11 and NCL-CBE1 were used for recognizing internal and external domains, respectively, of this protein. As indicated above, a number of different monoclonal and polyclonal antibodies have been used for determining the HER-2/neu status in breast cancer. A wide range of HER-2/neu overexpression rates has been reported in different studies, varying from 9–60%. This range resulted from using a wide variety of antibodies and/or variations in preparation techniques, scoring criteria, and stage of cancer. The HercepTest containing rabbitpolyclonal antibody A0485 (Dako) is superior to other antibodies. Recently, this antibody was compared with five monclonal antibodies (9G6, 3B5, CB11, TAB 250, GSF-HER 2) and one polyclonal antibody (A8010), using formalin-fixed, paraffin-embedded archival invasive breast cancer tissues (Lebeau et al., 2001). This study has confirmed the superiority of A0485 over other antibodies. Another study also indicates the superiority of A0485 over polyclonal antibody available from Oncor, Inc. (Gaithersburg, MD) (Maia, 1999). In conclusion, the type of antibody used will affect the results of HER-2/neu protein immunohistochemistry. Figure 12.3 shows the immunostaining of HER-2 oncoprotein in invasive breast carcinoma, using monoclonal anti-c-erbB-2 antibody. Table 12.2 shows immunohistochemical localization of this protein in different carcinomas in various tissue and organ types.

HERCEPTIN (TRASTUZUMAB)
A large body of evidence in the literature supports the idea that antibodies directed against HER-2 can inhibit the growth of HER-2–expressing tumors through several mechanisms, including antibody-dependent, cell toxicity–mediated inhibition of HER-2 signal transduction. A number of murine monoclonal antibodies against the extracellular domain of the HER-2 protein have been found to inhibit the proliferation of human cancer cells that overexpress HER-2 both in vitro and in vivo (Hudziak et al., 1989; Shepard et al., 1991). To minimize immunogenicity, the antigen binding region of one of the more effective antibodies (Herceptin, Ab4D5) was fused to the framework region of human IgG and tested against breast cancer cells that overexpress HER-2 in vivo and in vitro (Carter et al., 1992; Pietras et al., 1994). It is well established that Herceptin is superior to other antibodies (e.g., polyconal antibody, Oncor, Inc.) and is called recombinant humanized anti-HER-2 antibody. Although Herceptin inhibits tumor growth when used alone, it has synergistic effects when used in combination with chemotherapy. Phase 1 clinical trials demonstrated that Herceptin is safe and confined to the tumor. However, phase 2 trials indicated that the efficacy of the antibody is superior when given with chemotherapy (Pegram et al., 1998). Phase 3 trials have indicated that Herceptin, when added to conventional chemotherapy, can benefit patients with metastatic breast cancer that overexpresses HER-2, prolonging relapse and overall survival (Slamon et al., 2001). Similarly, extensive studies by Menden et al. (2001) support the weekly Docetaxel

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and Herceptin combination therapy for women with HER-2 overexpression metastatic breast cancer. This combined treatment is thought to be well tolerated with significant antitumor activity. However, a most troubling adverse effect of this antibody is cardiac dysfunction, especially when given concurrently with an anthracycline. The mechanism of the cardiotoxicity of Herceptin is not known. Therefore, great caution is required in using this antibody. Additional clinical trials are needed to further evaluate and confirm the usefulness of Herceptin when used in combination with chemotherapy.

HERCEPTEST
The immunohistochemistry of estrogen receptors and progesterone receptors provides valuable information, aiding in the selection of breast cancer patients for endocrine treatment. However, consensus on the effectiveness of this approach is lacking. Immunostaining of HER-2/neu gene product (HER-2), on the other hand, is an effective prognostic indicator in patients with breast cancer. In 1998 the U.S. Food and Drug Administration (FDA) and the Health Protection Branch of Canada approved HER-2 use for the HercepTest for immunohistochemical detection of HER-2 overexpression in breast tumors of patients who are being considered for Herceptin (trastuzumab) therapy. Unlike many chemotherapeutic agents that destroy any population of dividing cells, Herceptin targets a specific molecular abnormality which is absent on nonneoplastic cells. Dako’s immunohistochemical assay is the only FDA-approved protocol for detecting HER-2 oncoprotein overexpression. Dako’s kit is used for analyzing this oncoprotein in sections of paraffin-embedded breast tumors using immunohistochemistry or FISH. These two assays determine the patient’s HER-2/neu status. Immunostaining of the HER-2/neu gene product and the detection of oncogene amplification demonstrate good correlation. Gene amplification detected by FISH is 92% concordant with immunohistochemical detection of overexpression of the oncoprotein (Persons et al., 1997); however, the percentage may be higher for HER 3+ cases. Although the two methods mentioned above are viable options, they should not be applied independently of each other. A tested, standardized, and well-controlled immunohistochemical assay should serve as the first test. It is simple to carry out and quite reliable for the two extreme ends of the staining spectrum (9 and 3+) (Hendricks, 2000). The 2+ cases (weak staining in at least 10% of neoplastic cells) can in turn be subjected to FISH analysis to confirm the presence of an altered HER-2/neu gene. The results of the HercepTest are interpreted in a semiquantitative manner, with scores ranging from 0–3+, reflecting the intensity of the staining reaction (Table 12.3). Thus, Herceptin therapy is based on the difference between faint and weak staining. Contrary to some other immunohistochemical assays, which are based on the percentage of positive cells, the HercepTest immunohistochemical assay is based on both the intensity and extent of staining. The subjective nature of judging stain intensity invites bias even when positive controls are included in the study (Hendricks, 2000). For the HercepTest to be valid, it must be performed exactly according to the manufacturer’s directions. The tissue must be fixed in 10% neutral buffered formalin or Bouin’s solution. Sections of paraffin-embedded tissues should be cut thick and processed in a standard tissue processor. Epitope retrieval should be carried out in a water bath instead of in a microwave oven. Water is used because it provides more uniform and

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consistent heating than do other heating methods. Any variation in the manufacturer’s instructions can lead to variability in results. Indeed, interobserver and interlaboratory variability of the interpretation of immunohistochemical staining using the HercepTest is not uncommon for HER 1 + cases, because in these cases it is not clinically relevant. In other words, scores of 0 and 1 + are considered negative and indicate probable nonresponsiveness to Herceptin. The variability is applicable for HER 2+ and 3+ cases. The scoring systems and cutoffs used in different studies may vary. Therefore, there is a need for optimizing the staining evaluation system. This variability can be significantly minimized by quantifying HER-2 expression by image analysis (Press et al., 1993). In a recent elegant study, Hatanaka et al. (2001) have quantified the levels of HER-2 protein expression in breast cancer using an image analyzing system and applied this system for optimizing the interpretation of the HercepTest. By converting the quantitatively extracted data into a scoring system based on the criteria, the outcome demonstrates a strong concordance with the scoring data obtained from immunostaining. Figure 12.4 (Plate 6) shows the image analysis of breast carcinoma immunostained with the HercepTest. In this system, brown and blue signals are extracted and replaced with artificial green images. A problem with the HercepTest arises when it is used at high altitudes. One of the requirements of this test is that the slides must be heated in Epitope Retrieval Solution (Dako) in a water bath at 95°C for 40 min. While this temperature can be easily obtained at sea level, it is difficult to achieve at altitudes above 5,000 feet. At these altitudes, water boils rapidly below 95°C as a result of air pressure on the boiling point. The use of boiling water does not conform to the manufacturer’s directions. Various solutions to this problem include extending the duration of heating to 50 min at 90°C or placing a metal lid over the water bath. Any adjustment in the heating step should be assessed using the 1 + control cell line included in the HercepTest kit. Ruegg and Lupfer (2001) have summarized various solutions to this problem.

Controls and Scoring System
For the negative control, primary antibody is replaced with an irrelevant, isotypematched antibody. This step controls nonspecific binding of the secondary antibody. The

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positive control slides consist of sections of cell blocks of the three breast cancer cell lines SKBR3, MDA-MB-175, and MDA-MB-231, which express 2.4 million, 92,000, and 22,000 HER-2 receptor molecules, respectively, by Scatchard analysis (Koeppen et al., 2001). These receptor numbers correspond to immunohistochemical HER-2 scores of 3, 1, and 0. These scores are defined by a lack of staining or membranous staining in less than 10% of the cells (0), incomplete membranous staining in more than 10% of the cells (score 1), and complete membranous staining of strong intensity in more than 10% of the cells (score 3). Scores of 2 or higher indicate HER-2 overexpression. A score of 2 or higher is generally considered to be staining of 20–35% of the cells. Only membranous staining should be considered as positive. The incidence of HER-2 overexpression varies considerably in different tumors. Table 12.4 shows an average score of HER-2 overexpression in representative human solid tumors commonly evaluated in clinical practice. The specimens in this study consisted of resections of primary tumors. Locally advanced and metastatic lesions of the same tumor types may show significantly different incidences of HER-2 overexpression. Infiltrating ductal carcinoma is one of the few tumor categories for which the incidence of HER-2 overexpression is consistent in most studies. These data were obtained using a single, standardized immunohistochemical method (Herceptin).

IMMUNOSTAINING OF HER-2 PROTEIN USING HERCEPTEST
Breast tumor tissues are fixed with formalin for 24 hr and embedded in paraffin, and sections are deparaffinized according to standard procedures. They are heated in 0.1 mM sodium citrate buffer (pH 6.0) in a water bath for 40 min at 95°C. After cooling for 20 min, the sections are treated with 0.3% hydrogen peroxide containing 15 mM sodium azide for

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10 min to block endogenous peroxidase activity. They are thoroughly rinsed in a washing buffer (50 mM Tris-HCI buffered saline [pH 7.6] containing a detergent), incubated with anti-HER-2/neu antibody for 30 min, and washed three times with the washing buffer. The antibody, bound to HER-2 protein, is detected by incubation (for 30 min) with the dextran polymer reagent conjugated with peroxidase and secondary antibody. Following washing with the washing buffer, color development is achieved with DAB for 10 min in an automatic stainer. As controls, slides containing three cell lines showing different HER-2 protein expression are included in the staining protocol. This step is used to confirm validation of the staining results. Semiquantitative analysis is carried out based on a score of 0 (no staining or membrane staining on less than 10% of tumor cells), 1+ (a faint perceptible staining), 2+ (a weak to moderate staining of the entire membrane), and 3+ (a strong staining of the entire membrane). Scores of 1+ to 3+ are also essential to be positive for more than 10% of tumor cells. Scores of 0 or 1+ are negative for HER-2 protein overexpression, while scores of 2+ and 3+ are weak positive and strong positive, respectively. Only membrane staining is evaluated. Quantitative analysis of the HER-2/neu expression is calculated using a color video camera with image processing software. The three images for analysis are picked up from tumor lesions that exhibit predominant and typical features microscopically in each case (Hatanaka et al., 2001). The quantitative labeling index for assessment is calculated by the ratio of brown membranous staining area stained with DAB to round blue areas stained with hematoxylin in consideration of tumor cell density in selected image. The extraction of a brown or blue signal is carried out with specific protocols based on RGB color parameter, selected automatically in accordance with the protocols, and highlighted as artificial green images (Fig. 12.4/Plate 6).

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