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CONTENTS i
FOURTEENTH EDITION
Junqueira’s
Basic Histology T E X T A N D AT L A S
Anthony L. Mescher, PhD

Professor of Anatomy and Cell Biology
Indiana University School of Medicine
Bloomington, Indiana
New York Chicago San Francisco Athens London Madrid Mexico City
Milan New Delhi Singapore Sydney Toronto
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Contents
KEY FEATURESâ•‡ VIâ•‡ |â•‡ PREFACEâ•‡IXâ•‡|â•‡ACKNOWLEDGMENTSâ•‡XI
1 Histology & Its Methods 5 Connective Tissueâ•‡ 96

of Studyâ•‡ 1 Cells of Connective Tissueâ•‡ 96
Preparation of Tissues for Studyâ•‡ 1 Fibersâ•‡103
Light Microscopyâ•‡ 4 Ground Substanceâ•‡ 111
Electron Microscopyâ•‡ 8 Types of Connective Tissueâ•‡ 114
Autoradiographyâ•‡9 Summary of Key Pointsâ•‡ 119
Cell & Tissue Cultureâ•‡ 10 Assess Your Knowledgeâ•‡ 120
Enzyme Histochemistryâ•‡ 10
Visualizing Specific Moleculesâ•‡ 10 6 Adipose Tissueâ•‡ 122
Interpretation of Structures in Tissue White Adipose Tissueâ•‡ 122
Sectionsâ•‡14 Brown Adipose Tissueâ•‡ 126
Summary of Key Pointsâ•‡ 15 Summary of Key Pointsâ•‡ 127
Assess Your Knowledgeâ•‡ 16 Assess Your Knowledgeâ•‡ 128
2 The Cytoplasmâ•‡ 17 7 Cartilageâ•‡129

Cell Differentiationâ•‡ 17 Hyaline Cartilageâ•‡ 129
The Plasma Membraneâ•‡ 17 Elastic Cartilageâ•‡ 133
Cytoplasmic Organellesâ•‡ 27 Fibrocartilageâ•‡134
The Cytoskeletonâ•‡ 42 Cartilage Formation, Growth, & Repairâ•‡ 134
Inclusionsâ•‡47 Summary of Key Pointsâ•‡ 136
Summary of Key Pointsâ•‡ 51 Assess Your Knowledgeâ•‡ 136
Assess Your Knowledgeâ•‡ 52
8 Boneâ•‡138
3 The Nucleusâ•‡ 53 Bone Cellsâ•‡ 138
Components of the Nucleusâ•‡ 53 Bone Matrixâ•‡ 143
The Cell Cycleâ•‡ 58 Periosteum & Endosteumâ•‡ 143
Mitosisâ•‡61 Types of Boneâ•‡ 143
Stem Cells & Tissue Renewalâ•‡ 65 Osteogenesisâ•‡148
Meiosisâ•‡65 Bone Remodeling & Repairâ•‡ 152
Apoptosisâ•‡67 Metabolic Role of Boneâ•‡ 153
Summary of Key Pointsâ•‡ 69 Jointsâ•‡155
Assess Your Knowledgeâ•‡ 70 Summary of Key Pointsâ•‡ 158
4 Epithelial Tissueâ•‡ 71
Characteristic Features of Epithelial Cellsâ•‡ 72 9 Nerve Tissue & the Nervous
Specializations of the Apical Cell Surfaceâ•‡ 77 Systemâ•‡161
Types of Epitheliaâ•‡ 80 Development of Nerve Tissueâ•‡ 161
Transport Across Epitheliaâ•‡ 88 Neuronsâ•‡163
Renewal of Epithelial Cellsâ•‡ 88 Glial Cells & Neuronal Activityâ•‡ 168
Summary of Key Pointsâ•‡ 90 Central Nervous Systemâ•‡ 175
Assess Your Knowledgeâ•‡ 93 Peripheral Nervous Systemâ•‡ 182
iii
iv CONTENTS
Neural Plasticity & Regenerationâ•‡ 187 15 Digestive Tractâ•‡ 295

Summary of Key Pointsâ•‡ 190
General Structure of the Digestive Tractâ•‡ 295
Oral Cavityâ•‡ 298
Esophagusâ•‡305
10 Muscle Tissueâ•‡ 193 Stomachâ•‡307
Skeletal Muscleâ•‡ 193 Small Intestineâ•‡ 314
Cardiac Muscleâ•‡ 207 Large Intestineâ•‡ 318
Smooth Muscleâ•‡ 208 Summary of Key Pointsâ•‡ 326
Regeneration of Muscle Tissueâ•‡ 213 Assess Your Knowledgeâ•‡ 328
Assess Your Knowledgeâ•‡ 214 16 Organs Associated with the Digestive
Tractâ•‡329
11 The Circulatory Systemâ•‡ 215 Salivary Glandsâ•‡ 329
Heartâ•‡215 Pancreasâ•‡332
Tissues of the Vascular Wallâ•‡ 219 Liverâ•‡335
Vasculatureâ•‡220 Biliary Tract & Gallbladderâ•‡ 345
Lymphatic Vascular Systemâ•‡ 231 Summary of Key Pointsâ•‡ 346
17 The Respiratory Systemâ•‡ 349

12 Bloodâ•‡ 237 Nasal Cavitiesâ•‡ 349
Composition of Plasmaâ•‡ 237 Pharynxâ•‡352
Blood Cellsâ•‡ 239 Larynxâ•‡352
Summary of Key Pointsâ•‡ 250 Tracheaâ•‡354
Assess Your Knowledgeâ•‡ 252 Bronchial Tree & Lungâ•‡ 354
Lung Vasculature & Nervesâ•‡ 366
13 Hemopoiesisâ•‡ 254 Pleural Membranesâ•‡ 368
Stem Cells, Growth Factors, & Differentiationâ•‡ 254 Respiratory Movementsâ•‡ 368
Bone Marrowâ•‡ 255 Summary of Key Pointsâ•‡ 369
Maturation of Erythrocytesâ•‡ 258 Assess Your Knowledgeâ•‡ 369
Maturation of Granulocytesâ•‡ 260
Maturation of Agranulocytesâ•‡ 263 18 Skinâ•‡ 371
Origin of Plateletsâ•‡ 263 Epidermisâ•‡372
Summary of Key Pointsâ•‡ 265 Dermisâ•‡378
Assess Your Knowledgeâ•‡ 265 Subcutaneous Tissueâ•‡ 381
Sensory Receptorsâ•‡ 381
14 The Immune System & Lymphoid Hairâ•‡383
Organsâ•‡267 Nailsâ•‡384
Innate & Adaptive Immunityâ•‡ 267 Skin Glandsâ•‡ 385
Cytokinesâ•‡269 Skin Repairâ•‡ 388
Antigens & Antibodiesâ•‡ 270 Summary of Key Pointsâ•‡ 391
Antigen Presentationâ•‡ 271 Assess Your Knowledgeâ•‡ 391
Cells of Adaptive Immunityâ•‡ 273
Thymusâ•‡276 19 The Urinary Systemâ•‡ 393
Mucosa-Associated Lymphoid Tissueâ•‡ 281 Kidneysâ•‡393
Lymph Nodesâ•‡ 282 Blood Circulationâ•‡ 394
Spleenâ•‡286 Renal Function: Filtration, Secretion, &
Summary of Key Pointsâ•‡ 293 Reabsorptionâ•‡395
Assess Your Knowledgeâ•‡ 294 Ureters, Bladder, & Urethraâ•‡ 406
CONTENTS v
Summary of Key Pointsâ•‡ 411 22 The Female Reproductive Systemâ•‡ 460

Ovariesâ•‡460
Uterine Tubesâ•‡ 470
20 Endocrine Glandsâ•‡ 413 Major Events of Fertilizationâ•‡ 471
Pituitary Gland (Hypophysis)â•‡ 413 Uterusâ•‡471
Adrenal Glandsâ•‡ 423 Embryonic Implantation, Decidua, & the Placentaâ•‡ 478
Pancreatic Isletsâ•‡ 427 Cervixâ•‡482
Diffuse Neuroendocrine Systemâ•‡ 429 Vaginaâ•‡483
Thyroid Glandâ•‡ 429 External Genitaliaâ•‡ 483
Parathyroid Glandsâ•‡ 432 Mammary Glandsâ•‡ 483
Pineal Glandâ•‡ 434 Summary of Key Pointsâ•‡ 488
23 The Eye & Ear: Special Sense
21 The Male Reproductive Organsâ•‡490
Systemâ•‡439 Eyes: The Photoreceptor Systemâ•‡ 490
Testesâ•‡439 Ears: The Vestibuloauditory Systemâ•‡ 509
Intratesticular Ductsâ•‡ 449 Summary of Key Pointsâ•‡ 522
Excretory Genital Ductsâ•‡ 450 Assess Your Knowledgeâ•‡ 522
Accessory Glandsâ•‡ 451
Penisâ•‡456 APPENDIXâ•‡525
FIGURE CREDITSâ•‡527
INDEXâ•‡529
Preface
With this 14th edition, Junqueira’s Basic Histology continues as throughout the book as needed, and again make up a complete
the preeminent source of concise yet thorough information atlas of cell, tissue, and organ structures fully compatible
on human tissue structure and function. For nearly 45 years with the students’ own collection of glass or digital slides. A
this educational resource has met the needs of learners for a virtual microscope with over 150 slides of all human tissues
well-organized and concise presentation of cell biology and and organs is available: http://medsci.indiana.edu/junqueira/
histology that integrates the material with that of biochemistry, virtual/junqueira.htm.
immunology, endocrinology, and physiology and provides As with the previous edition, the book facilitates learning
an excellent foundation for subsequent studies in pathology. by its organization:
The text is prepared specifically for students of medicine
and other health-related professions, as well as for advanced
■ An opening chapter reviews the histological techniques
that allow understanding of cell and tissue structure.
undergraduate courses in tissue biology. As a result of its value
and appeal to students and instructors alike, Junqueira’s Basic
■ Two chapters then summarize the structural and
functional organization of human cell biology,
Histology has been translated into a dozen different languages
presenting the cytoplasm and nucleus separately.
and is used by medical students throughout the world.
This edition now includes with each chapter a set of
■ The next seven chapters cover the four basic tissues that
make up our organs: epithelia, connective tissue (and its
multiple-choice Self-Test Questions that allow readers to assess
major sub-types), nervous tissue, and muscle.
their comprehension and knowledge of important material in
that chapter. At least a few questions in each set utilize clinical
■ Remaining chapters explain the organization and
functional significance of these tissues in each of
vignettes or cases to provide context for framing the medical
the body’s organ systems, closing with up-to-date
relevance of concepts in basic science, as recommended by
consideration of cells in the eye and ear.
the US National Board of Medical Examiners. As with the
last edition, each chapter also includes a Summary of Key For additional review of what’s been learned or to
Points designed to guide the students concerning what is assist rapid assimilation of the material in Junqueira’s Basic
clearly important and what is less so. Summary Tables in Histology, McGraw-Hill has published a set of 200 full-color
each chapter organize and condense important information, Basic Histology Flash Cards, Anthony Mescher author. Each
further facilitating efficient learning. card includes images of key structures to identify, a summary
Each chapter has been revised and shortened, while of important facts about those structures, and a clinical
coverage of specific topics has been expanded as needed. Study comment. This valuable learning aid is available as a set of
is facilitated by modern page design. Inserted throughout each actual cards from Amazon.com, or as an app for smart phones
chapter are more numerous, short paragraphs that indicate how or tablets from the online App Store.
the information presented can be used medically and which With its proven strengths and the addition of new features,
emphasize the foundational relevance of the material learned. I am confident that Junqueira’s Basic Histology will continue
The art and other figures are presented in each chapter, as one of the most valuable and most widely read educational
with the goal to simplify learning and integration with resources in histology. Users are invited to provide feedback
related material. The McGraw-Hill medical illustrations, now to the author with regard to any aspect of the book’s features.
used throughout the text and supplemented by numerous
animations in the electronic version of the text, are the Anthony L. Mescher
most useful, thorough, and attractive of any similar medical Indiana University School of Medicine
textbook. Electron and light micrographs have been replaced mescher@indiana.edu
ix
Acknowledgments
I wish to thank the students at Indiana University School of these valuable resources is gratefully acknowledged. Students are
Medicine and the undergraduates at Indiana University with referred to those review books for hundreds of additional self-
whom I have studied histology and cell biology for over 30 years assessment questions.
and from whom I have learned much about presenting basic I am also grateful to my colleagues and reviewers from
concepts most effectively. Their input has greatly helped in the throughout the world who provided specialized expertise
task of maintaining and updating the presentations in this classic or original photographs, which are also acknowledged in
textbook. As with the last edition the help of Sue Childress and figure captions. I thank those professors and students in the
Dr. Mark Braun was invaluable in slide preparation and the United States, as well as Argentina, Canada, Iran, Ireland, Italy,
virtual microscope for human histology respectively. Pakistan, and Syria, who provided useful suggestions that
A major change in this edition is the inclusion of self- have improved the new edition of Junqueira’s Basic Histology.
assessment questions with each topic/chapter. Many of these Finally, I am pleased to acknowledge the help and collegiality
questions were used in my courses, but others are taken or provided by the staff of McGraw-Hill, especially editors
modified from a few of the many excellent review books published Michael Weitz and Brian Kearns, whose work made possible
by McGraw-Hill/Lange for students preparing to take the U.S. publication of this 14th edition of Junqueira’s Basic Histology.
Medical Licensing Examination. These include Histology and Cell
Biology: Examination and Board Review, by Douglas Paulsen; Anthony L. Mescher
USMLE Road Map: Histology, by Harold Sheedlo; and Anatomy,
Indiana University School of Medicine
Histology, & Cell Biology: PreTest Self-Assessment & Review, by
Robert Klein and George Enders. The use here of questions from mescher@indiana.edu
xi
C H A P T E R
1
PREPARATION OF TISSUES FOR STUDY
Histology & Its
Methods of Study
1 AUTORADIOGRAPHY 9
Fixation 1 CELL & TISSUE CULTURE 10
Embedding & Sectioning 3
ENZYME HISTOCHEMISTRY 10
Staining 3
LIGHT MICROSCOPY 4 VISUALIZING SPECIFIC MOLECULES 10
Bright-Field Microscopy 4 Immunohistochemistry 11
Fluorescence Microscopy 5 Hybridization Techniques 12
Phase-Contrast Microscopy 5 INTERPRETATION OF STRUCTURES IN TISSUE
Confocal Microscopy 5 SECTIONS 14
Polarizing Microscopy 7 SUMMARY OF KEY POINTS 15
ELECTRON MICROSCOPY 8 ASSESS YOUR KNOWLEDGE 16
Transmission Electron Microscopy 8
Scanning Electron Microscopy 9
H istology is the study of the tissues of the body and

how these tissues are arranged to constitute organs.
This subject involves all aspects of tissue biology, with
the focus on how cells’ structure and arrangement optimize
functions specific to each organ.
a better knowledge of tissue biology. Familiarity with the tools
and methods of any branch of science is essential for a proper
understanding of the subject. This chapter reviews common
methods used to study cells and tissues, focusing on micro-
scopic approaches.
Tissues have two interacting components: cells and
extracellular matrix (ECM). The ECM consists of many kinds
of macromolecules, most of which form complex structures,
such as collagen fibrils. The ECM supports the cells and
›â•ºPREPARATION OF TISSUES
contains the fluid transporting nutrients to the cells, and FOR STUDY
carrying away their wastes and secretory products. Cells The most common procedure used in histologic research is
produce the ECM locally and are in turn strongly influenced the preparation of tissue slices or “sections” that can be exam-
by matrix molecules. Many matrix components bind to spe- ined visually with transmitted light. Because most tissues and
cific cell surface receptors that span the cell membranes and organs are too thick for light to pass through, thin translu-
connect to structural components inside the cells, forming a cent sections are cut from them and placed on glass slides for
continuum in which cells and the ECM function together in microscopic examination of the internal structures.
a well-coordinated manner. The ideal microscopic preparation is preserved so that the
During development, cells and their associated matrix tissue on the slide has the same structural features it had in the
become functionally specialized and give rise to fundamen- body. However, this is often not feasible because the prepara-
tal types of tissues with characteristic structural features. tion process can remove cellular lipid, with slight distortions
Organs are formed by an orderly combination of these tissues, of cell structure. The basic steps used in tissue preparation for
and their precise arrangement allows the functioning of each light microscopy are shown in Figure 1–1.
organ and of the organism as a whole.
The small size of cells and matrix components makes his-
tology dependent on the use of microscopes and molecular Fixation
methods of study. Advances in biochemistry, molecular biol- To preserve tissue structure and prevent degradation by
ogy, physiology, immunology, and pathology are essential for enzymes released from the cells or microorganisms, pieces of
1
2 CHAPTER 1â•… ■â•… Histology & Its Methods of Study
FIGURE 1–1â•‡ Sectioning fixed and embedded tissue.
52°- 60°C
(a) Fixation Dehydration Clearing Infiltration Embedding
Drive wheel
Block holder
Paraffin block
Tissue
Steel knife
Most tissues studied histologically are prepared as shown, with Similar steps are used in preparing tissue for transmission elec-
this sequence of steps (a): tron microscopy (TEM), except special fixatives and dehydrating
solutions are used with smaller tissue samples and embedding
■âŒ€ Fixation: Small pieces of tissue are placed in solutions of
involves epoxy resins which become harder than paraffin to allow
chemicals that cross-link proteins and inactivate degradative
very thin sectioning.
enzymes, which preserves cell and tissue structure.
■âŒ€ Dehydration: The tissue is transferred through a series of (b) A microtome is used for sectioning paraffin-embedded tissues
increasingly concentrated alcohol solutions, ending in 100%, for light microscopy. The trimmed tissue specimen is mounted
which removes all water. in the paraffin block holder, and each turn of the drive wheel by
■âŒ€ Clearing: Alcohol is removed in organic solvents in which the histologist advances the holder a controlled distance, gener-
both alcohol and paraffin are miscible. ally from 1 to 10 μm. After each forward move, the tissue block
■âŒ€ Infiltration: The tissue is then placed in melted paraffin until it passes over the steel knife edge and a section is cut at a thickness
becomes completely infiltrated with this substance. equal to the distance the block advanced. The paraffin sections
■âŒ€ Embedding: The paraffin-infiltrated tissue is placed in a small are placed on glass slides and allowed to adhere, deparaffinized,
mold with melted paraffin and allowed to harden. and stained for light microscope study. For TEM, sections less than
■âŒ€ Trimming: The resulting paraffin block is trimmed to expose 1 μm thick are prepared from resin-embedded cells using an
the tissue for sectioning (slicing) on a microtome. ultramicrotome with a glass or diamond knife.
organs are placed as soon as possible after removal from the microscopy, react with the amine groups (NH2) of proteins,
body in solutions of stabilizing or cross-linking compounds preventing their degradation by common proteases. Glutaral-
called fixatives. Because a fixative must fully diffuse through dehyde also cross-links adjacent proteins, reinforcing cell and
the tissues to preserve all cells, tissues are usually cut into small ECM structures.
fragments before fixation to facilitate penetration. To improve Electron microscopy provides much greater magnification
cell preservation in large organs fixatives are often introduced and resolution of very small cellular structures and fixation
via blood vessels, with vascular perfusion allowing fixation must be done very carefully to preserve additional “ultra-
rapidly throughout the tissues. structural” detail. Typically in such studies glutaraldehyde-
One widely used fixative for light microscopy is forma- treated tissue is then immersed in buffered osmium
lin, a buffered isotonic solution of 37% formaldehyde. Both tetroxide, which preserves (and stains) cellular lipids as well
this compound and glutaraldehyde, a fixative used for electron as proteins.
Preparation of Tissues for Study 3
Embedding & Sectioning Staining
C H A P T E R
To permit thin sectioning fixed tissues are infiltrated and Most cells and extracellular material are completely color-
embedded in a material that imparts a firm consistency. less, and to be studied microscopically tissue sections must
Embedding materials include paraffin, used routinely for light be stained (dyed). Methods of staining have been devised that
microscopy, and plastic resins, which are adapted for both make various tissue components not only conspicuous but also
light and electron microscopy. distinguishable from one another. Dyes stain material more or
Before infiltration with such media the fixed tissue must less selectively, often behaving like acidic or basic compounds
undergo dehydration by having its water extracted gradually and forming electrostatic (salt) linkages with ionizable radicals
1
by transfers through a series of increasing ethanol solutions, of macromolecules in tissues. Cell components such as nucleic
Histology & Its Methods of Study â•‡ ■â•‡ Preparation of Tissues for Study
ending in 100% ethanol. The ethanol is then replaced by an acids with a net negative charge (anionic) have an affinity for
organic solvent miscible with both alcohol and the embedding basic dyes and are termed basophilic; cationic components,
medium, a step referred to as clearing because infiltration with such as proteins with many ionized amino groups, stain more
the reagents used here gives the tissue a translucent appearance. readily with acidic dyes and are termed acidophilic.
The fully cleared tissue is then placed in melted paraffin Examples of basic dyes include toluidine blue, alcian blue,
in an oven at 52°-60°C, which evaporates the clearing solvent and methylene blue. Hematoxylin behaves like a basic dye,
and promotes infiltration of the tissue with paraffin, and then staining basophilic tissue components. The main tissue com-
embedded by allowing it to harden in a small container of ponents that ionize and react with basic dyes do so because of
paraffin at room temperature. Tissues to be embedded with acids in their composition (DNA, RNA, and glycosaminogly-
plastic resin are also dehydrated in ethanol and then infiltrated cans). Acid dyes (eg, eosin, orange G, and acid fuchsin) stain
with plastic solvents that harden when cross-linking polymer- the acidophilic components of tissues such as mitochondria,
izers are added. Plastic embedding avoids the higher tempera- secretory granules, and collagen.
tures needed with paraffin, which helps avoid tissue distortion. Of all staining methods, the simple combination of
The hardened block with tissue and surrounding embed- hematoxylin and eosin (H&E) is used most commonly.
ding medium is trimmed and placed for sectioning in an Hematoxylin stains DNA in the cell nucleus, RNA-rich por-
instrument called a microtome (Figure 1–1). Paraffin sections tions of the cytoplasm, and the matrix of cartilage, produc-
are typically cut at 3-10 μm thickness for light microscopy, but ing a dark blue or purple color. In contrast, eosin stains other
electron microscopy requires sections less than 1 μm thick. cytoplasmic structures and collagen pink (Figure 1–2a). Here
One micrometer (1 μm) equals 1/1000 of a millimeter (mm) eosin is considered a counterstain, which is usually a single
or 10–6 m. Other spatial units commonly used in microscopy dye applied separately to distinguish additional features of a
are the nanometer (1 nm = 0.001 μm = 10–6 mm = 10–9 m) and tissue. More complex procedures, such as trichrome stains (eg,
angstrom (1 Å = 0.1 nm or 10–4 μm). The sections are placed Masson trichrome), allow greater distinctions among various
on glass slides and stained for light microscopy or on metal extracellular tissue components.
grids for electron microscopic staining and examination. The periodic acid-Schiff (PAS) reaction utilizes the
hexose rings of polysaccharides and other carbohydrate-rich
› â•ºâ•º MEDICAL APPLICATION tissue structures and stains such macromolecules distinctly
purple or magenta. Figure 1–2b shows an example of cells with
Biopsies are tissue samples removed during surgery or rou- carbohydrate-rich areas well-stained by the PAS reaction. The
tine medical procedures. In the operating room, biopsies DNA of cell nuclei can be specifically stained using a modifica-
are fixed in vials of formalin for processing and microscopic tion of the PAS procedure called the Feulgen reaction.
analysis in a pathology laboratory. If results of such analyses Basophilic or PAS-positive material can be further identi-
are required before the medical procedure is completed, for fied by enzyme digestion, pretreatment of a tissue section with
example to know whether a growth is malignant before the an enzyme that specifically digests one substrate. For example,
patient is closed, a much more rapid processing method is pretreatment with ribonuclease will greatly reduce cytoplas-
used. The biopsy is rapidly frozen in liquid nitrogen, preserv- mic basophilia with little overall effect on the nucleus, indicat-
ing cell structures and making the tissue hard and ready for ing the importance of RNA for the cytoplasmic staining.
sectioning. A microtome called a cryostat in a cabinet at Lipid-rich structures of cells are revealed by avoiding the
subfreezing temperature is used to section the block with processing steps that remove lipids, such as treatment with
tissue, and the frozen sections are placed on slides for rapid heat and organic solvents, and staining with lipid-soluble
staining and microscopic examination by a pathologist. dyes such as Sudan black, which can be useful in diagnosis
Freezing of tissues is also effective in histochemical stud- of metabolic diseases that involve intracellular accumulations
ies of very sensitive enzymes or small molecules because of cholesterol, phospholipids, or glycolipids. Less common
freezing, unlike fixation, does not inactivate most enzymes. methods of staining can employ metal impregnation tech-
Finally, because clearing solvents often dissolve cell lipids in niques, typically using solutions of silver salts to visual certain
fixed tissues, frozen sections are also useful when structures ECM fibers and specific cellular elements in nervous tissue.
containing lipids are to be studied histologically. The Appendix lists important staining procedures used for
most of the light micrographs in this book.
FIGURE 1–2â•‡ Hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining.
G G
G
L
L
a b
Micrographs of epithelium lining the small intestine, (a) stained lumen, where projecting microvilli have a prominent layer of
with H&E, and (b) stained with the PAS reaction for glycoproteins. glycoproteins at the lumen (L) and in the mucin-rich secretory
With H&E, basophilic cell nuclei are stained purple while cyto- granules of goblet cells. Cell surface glycoproteins and mucin are
plasm stains pink. Cell regions with abundant oligosaccharides PAS-positive because of their high content of oligosaccharides
on glycoproteins, such as the ends of the cells at the lumen (L) and polysaccharides respectively. The PAS-stained tissue was
or the scattered mucus-secreting goblet cells (G), are poorly counterstained with hematoxylin to show the cell nuclei. (a. X400;
stained. With PAS, however, cell staining is most intense at the b. X300)
Slide preparation, from tissue fixation to observation (or ocular lens) further magnifying this image and projecting
with a light microscope, may take from 12 hours to 2½ days, it onto the viewer’s retina or a charge-coupled device (CCD)
depending on the size of the tissue, the embedding medium, highly sensitive to low light levels with a camera and monitor.
and the method of staining. The final step before microscopic The total magnification is obtained by multiplying the magni-
observation is mounting a protective glass coverslip on the fying power of the objective and ocular lenses.
slide with clear adhesive. The critical factor in obtaining a crisp, detailed image
with a light microscope is its resolving power, defined as the
smallest distance between two structures at which they can be
›â•ºLIGHT MICROSCOPY seen as separate objects. The maximal resolving power of the
light microscope is approximately 0.2 μm, which can permit
Conventional bright-field microscopy, as well as more special- clear images magnified 1000-1500 times. Objects smaller or
ized applications like fluorescence, phase-contrast, confocal, thinner than 0.2 μm (such as a single ribosome or cytoplasmic
and polarizing microscopy, are all based on the interaction of microfilament) cannot be distinguished with this instrument.
light with tissue components and are used to reveal and study Likewise, two structures such as mitochondria will be seen as
tissue features. only one object if they are separated by less than 0.2 μm. The
microscope’s resolving power determines the quality of the
Bright-Field Microscopy image, its clarity and richness of detail, and depends mainly on
With the bright-field microscope stained tissue is examined the quality of its objective lens. Magnification is of value only
with ordinary light passing through the preparation. As shown when accompanied by high resolution. Objective lenses pro-
in Figure 1–3, the microscope includes an optical system and viding higher magnification are designed to also have higher
mechanisms to move and focus the specimen. The optical resolving power. The eyepiece lens only enlarges the image
components are the condenser focusing light on the object obtained by the objective and does not improve resolution.
to be studied; the objective lens enlarging and projecting the Virtual microscopy, typically used for study of bright-
image of the object toward the observer; and the eyepiece field microscopic preparations, involves the conversion of a
Light Microscopy 5
FIGURE 1–3â•‡ Components and light path of a Fluorescence Microscopy
C H A P T E R
bright-field microscope. When certain cellular substances are irradiated by light of
a proper wavelength, they emit light with a longer wave-
Eyepiece Interpupillar
adjustment
length—a phenomenon called fluorescence. In fluores-
Binocular
tubes Head cence microscopy, tissue sections are usually irradiated with
ultraviolet (UV) light and the emission is in the visible portion
of the spectrum. The fluorescent substances appear bright on
Stand
a dark background. For fluorescent microscopy the instru-
1
Measuring
ment has a source of UV or other light and filters that select
Histology & Its Methods of Study â•‡ ■â•‡ Light Microscopy

graticule
Beamsplitter
rays of different wavelengths emitted by the substances to be
Revolving
nosepiece visualized.
Specimen Objective Fluorescent compounds with affinity for specific cell
holder macromolecules may be used as fluorescent stains. Acridine
Mechanical
stage
orange, which binds both DNA and RNA, is an example.
On/off
switch When observed in the fluorescence microscope, these nucleic
Condenser Illumination
intensity
acids emit slightly different fluorescence, allowing them to be
Field lens
control localized separately in cells (Figure 1–4a). Other compounds
Field such as DAPI and Hoechst stain specifically bind DNA and
diaphragm
Collector
are used to stain cell nuclei, emitting a characteristic blue fluo-
lens rescence under UV. Another important application of fluores-
cence microscopy is achieved by coupling compounds such as
X-Y
Base translation fluorescein to molecules that will specifically bind to certain
Tungsten mechanism
cellular components and thus allow the identification of these
halogen
lamp structures under the microscope (Figure 1–4b). Antibodies
labeled with fluorescent compounds are extremely important
Photograph of a bright-field light microscope showing its in immunohistologic staining. (See the section Visualizing
mechanical components and the pathway of light from the Specific Molecules.)
substage lamp to the eye of the observer. The optical system
has three sets of lenses:
■âŒ€ The condenser collects and focuses a cone of light that illu-
Phase-Contrast Microscopy
minates the tissue slide on the stage. Unstained cells and tissue sections, which are usually trans-
■âŒ€ Objective lenses enlarge and project the illuminated parent and colorless, can be studied with these modified
image of the object toward the eyepiece. Interchangeable
light microscopes. Cellular detail is normally difficult to see
objectives with different magnifications routinely used in
histology include X4 for observing a large area (field) of the in unstained tissues because all parts of the specimen have
tissue at low magnification; X10 for medium magnification roughly similar optical densities. Phase-contrast micros-
of a smaller field; and X40 for high magnification of more copy, however, uses a lens system that produces visible images
detailed areas. from transparent objects and, importantly, can be used with
■âŒ€ The two eyepieces or oculars magnify this image another
living, cultured cells (Figure 1–5).
X10 and project it to the viewer, yielding a total magnifica-
tion of X40, X100, or X400. Phase-contrast microscopy is based on the principle
that light changes its speed when passing through cellular
(Used with permission from Nikon Instruments.)
and extracellular structures with different refractive indices.
These changes are used by the phase-contrast system to cause
the structures to appear lighter or darker in relation to each
stained tissue preparation to high-resolution digital images other. Because they allow the examination of cells without
and permits study of tissues using a computer or other digi- fixation or staining, phase-contrast microscopes are promi-
tal device, without an actual stained slide or a microscope. In nent tools in all cell culture laboratories. A modification of
this technique regions of a glass-mounted specimen are cap- phase-contrast microscopy is differential interference
tured digitally in a grid-like pattern at multiple magnifications microscopy with Nomarski optics, which produces an image
using a specialized slide-scanning microscope and saved as of living cells with a more apparent three-dimensional (3D)
thousands of consecutive image files. Software then converts aspect (Figure 1–5c).
this dataset for storage on a server using a format that allows
access, visualization, and navigation of the original slide with
common web browsers or other devices. With advantages in Confocal Microscopy
cost and ease of use, virtual microscopy is rapidly replacing With a regular bright-field microscope, the beam of light is rel-
light microscopes and collections of glass slides in histology atively large and fills the specimen. Stray (excess) light reduces
laboratories for students. contrast within the image and compromises the resolving
FIGURE 1–4â•‡ Appearance of cells with fluorescent microscopy.
N R
a b
Components of cells are often stained with compounds visible by filaments show nuclei with blue fluorescence and actin filaments
fluorescence microscopy. stained green. Important information such as the greater density
(a) Acridine orange binds nucleic acids and causes DNA in cell of microfilaments at the cell periphery is readily apparent. (Both
nuclei (N) to emit yellow light and the RNA-rich cytoplasm (R) to X500)
appear orange in these cells of a kidney tubule. (Figure 1–4b, used with permission from Drs Claire E. Walczak
and Rania Rizk, Indiana University School of Medicine,
(b) Cultured cells stained with DAPI (4′,6-diamino-2-phenylindole) Bloomington.)
that binds DNA and with fluorescein-phalloidin that binds actin
FIGURE 1–5â•‡ Unstained cells’ appearance in three types of light microscopy.
a b c
Living neural crest cells growing in culture appear differently in-phase light differently and produce an image of these features
with various techniques of light microscopy. Here the same field in all the cells.
of unstained cells, including two differentiating pigment cells, is (c) Differential interference microscopy: Cellular details are
shown using three different methods (all X200): highlighted in a different manner using Nomarski optics. Phase-
(a) Bright-field microscopy: Without fixation and staining, only contrast microscopy, with or without differential interference, is
the two pigment cells can be seen. widely used to observe live cells grown in tissue culture.
(b) Phase-contrast microscopy: Cell boundaries, nuclei, and (Used with permission from Dr Sherry Rogers, Department of Cell
cytoplasmic structures with different refractive indices affect Biology and Physiology, University of New Mexico, Albuquerque, NM.)
Light Microscopy 7
the specimen allows them to be digitally reconstructed into a

FIGURE 1–6â•‡ Principle of confocal microscopy.
3D image.
C H A P T E R
Laser Polarizing Microscopy
Polarizing microscopy allows the recognition of stained or
unstained structures made of highly organized subunits.
When normal light passes through a polarizing filter, it exits
1
Scanner
vibrating in only one direction. If a second filter is placed in
Histology & Its Methods of Study â•‡ ■â•‡ Light Microscopy

the microscope above the first one, with its main axis per-
Detector pendicular to the first filter, no light passes through. If, how-
ever, tissue structures containing oriented macromolecules
are located between the two polarizing filters, their repeti-
tive structure rotates the axis of the light emerging from the
polarizer and they appear as bright structures against a dark
background (Figure 1–7). The ability to rotate the direction
of vibration of polarized light is called birefringence and is
Plate with
pinhole
Beam
splitter FIGURE 1–7â•‡ Tissue appearance with bright-field
and polarizing microscopy.
Lens
Other
Focal plane out-of-focus
Specimen planes
Although a very small spot of light originating from one plane

of the section crosses the pinhole and reaches the detector,
rays originating from other planes are blocked by the blind.
Thus, only one very thin plane of the specimen is focused at a a
time. The diagram shows the practical arrangement of a confo-
cal microscope. Light from a laser source hits the specimen and
is reflected. A beam splitter directs the reflected light to a pin-
hole and a detector. Light from components of the specimen
that are above or below the focused plane is blocked by the
blind. The laser scans the specimen so that a larger area of the
specimen can be observed.
power of the objective lens. Confocal microscopy (Figure 1–6)

avoids these problems and achieves high resolution and sharp
focus by using (1) a small point of high-intensity light, often
from a laser, and (2) a plate with a pinhole aperture in front of
the image detector. The point light source, the focal point of b
the lens, and the detector’s pinpoint aperture are all optically
conjugated or aligned to each other in the focal plane (confo-
Polarizing light microscopy produces an image only of material
cal), and unfocused light does not pass through the pinhole. having repetitive, periodic macromolecular structure; features
This greatly improves resolution of the object in focus and without such structure are not seen. Pieces of thin, unsec-
allows the localization of specimen components with much tioned mesentery were stained with red picrosirius, orcein, and
greater precision than with the bright-field microscope. hematoxylin, placed on slides and observed by bright-field (a)
Confocal microscopes include a computer-driven mirror and polarizing (b) microscopy.
system (the beam splitter) to move the point of illumination (a) With bright-field microscopy collagen fibers appear red,
with thin elastic fibers and cell nuclei darker. (X40)
across the specimen automatically and rapidly. Digital images
captured at many individual spots in a very thin plane of focus (b) With polarizing microscopy, only the collagen fibers are vis-
ible and these exhibit intense yellow or orange birefringence.
are used to produce an “optical section” of that plane. Creat- (a: X40; b: X100)
ing such optical sections at a series of focal planes through
a feature of crystalline substances or substances containing The wavelength in an electron beam is much shorter than that
highly oriented molecules, such as cellulose, collagen, micro- of light, allowing a 1000-fold increase in resolution.
tubules, and actin filaments.
The utility of all light microscopic methods is greatly Transmission Electron Microscopy
extended through the use of digital cameras. Many features The transmission electron microscope (TEM) is an imag-
of digitized histological images can be analyzed quantitatively ing system that permits resolution around 3 nm. This high
using appropriate software. Such images can also be enhanced resolution allows isolated particles magnified as much as
to allow objects not directly visible through the eyepieces to be 400,000 times to be viewed in detail. Very thin (40-90 nm),
examined on a monitor. resin-embedded tissue sections are typically studied by TEM
at magnifications up to approximately 120,000 times.
›â•ºELECTRON MICROSCOPY Figure 1–8a indicates the components of a TEM and the
basic principles of its operation: a beam of electrons focused
Transmission and scanning electron microscopes are based on using electromagnetic “lenses” passes through the tissue sec-
the interaction of tissue components with beams of electrons. tion to produce an image with black, white, and intermediate
FIGURE 1–8â•‡ Electron microscopes.
Electron gun Electron gun

Cathode Cathode
3 mm
Anode Anode
Copper grid
Condensor lens with three sections Lens
Specimen Column
Objective lens holder Lens
Column Scanner
Intermediate lens Electron detector
TEM image Lens SEM image

Projector lens
Image on viewing
screen Specimen
Electron detector
with CCD camera
(a) Transmission electron microscope (b) Scanning electron microscope
Electron microscopes are large instruments generally housed in a In a TEM image areas of the specimen through which electrons
specialized EM facility. passed appear bright (electron lucent), while denser areas or
(a) Schematic view of the major components of a transmission elec- those that bind heavy metal ions during specimen preparation
tron microscope (TEM), which is configured rather like an upside- absorb or deflect electrons and appear darker (electron dense).
down light microscope. With the microscope column in a vacuum, a Such images are therefore always black, white, and shades of gray.
metallic (usually tungsten) filament (cathode) at the top emits elec- (b) The scanning electron microscope (SEM) has many similarities
trons that travel to an anode with an accelerating voltage between to a TEM. However, here the focused electron beam does not pass
60 and 120 kV. Electrons passing through a hole in the anode form a through the specimen, but rather is moved sequentially (scanned)
beam that is focused electromagnetically by circular electric coils from point to point across its surface similar to the way an electron
in a manner analogous to the effect of optical lenses on light. beam is scanned across a television tube or screen. For SEM speci-
The first lens is a condenser focusing the beam on the sec- mens are coated with metal atoms with which the electron beam
tion. Some electrons interact with atoms in the section, being interacts, producing reflected electrons and newly emitted secondary
absorbed or scattered to different extents, while others are simply electrons. All of these are captured by a detector and transmitted to
transmitted through the specimen with no interaction. Electrons amplifiers and processed to produce a black-and-white image on the
reaching the objective lens form an image that is then magnified monitor. The SEM shows only surface views of the coated specimen
and finally projected on a fluorescent screen or a charge-coupled but with a striking 3D, shadowed quality. The inside of organs or cells
device (CCD) monitor and camera. can be analyzed after sectioning to expose their internal surfaces.
Autoradiography 9
shades of gray regions. These regions of an electron micro- layer of heavy metal (often gold) which reflects electrons in
graph correspond to tissue areas through which electrons a beam scanning the specimen. The reflected electrons are
C H A P T E R
passed readily (appearing brighter or electron-lucent) and captured by a detector, producing signals that are processed
areas where electrons were absorbed or deflected (appearing to produce a black-and-white image. SEM images are usually
darker or more electron-dense). To improve contrast and reso- easy to interpret because they present a three-dimensional
lution in TEM, compounds with heavy metal ions are often view that appears to be illuminated in the same way that large
added to the fixative or dehydrating solutions used for tissue objects are seen with highlights and shadows caused by light.
preparation. These include osmium tetroxide, lead citrate,
1
and uranyl compounds, which bind cellular macromolecules,
›â•ºAUTORADIOGRAPHY
Histology & Its Methods of Study â•‡ ■â•‡Autoradiography

increasing their electron density and visibility.
Cryofracture and freeze etching are techniques that
Microscopic autoradiography is a method of localizing
allow TEM study of cells without fixation or embedding and
newly synthesized macromolecules in cells or tissue sections.
have been particularly useful in the study of membrane struc-
Radioactively labeled metabolites (nucleotides, amino acids,
ture. In these methods very small tissue specimens are rap-
sugars) provided to the living cells are incorporated into spe-
idly frozen in liquid nitrogen and then cut or fractured with a
cific macromolecules (DNA, RNA, protein, glycoproteins, and
knife. A replica of the frozen exposed surface is produced in a
polysaccharides) and emit weak radiation that is restricted
vacuum by applying thin coats of vaporized platinum or other
to those regions where the molecules are located. Slides with
metal atoms. After removal of the organic material, the replica
radiolabeled cells or tissue sections are coated in a darkroom
of the cut surface can be examined by TEM. With membranes
with photographic emulsion in which silver bromide crystals
the random fracture planes often split the lipid bilayers, expos-
ing protein components whose size, shape, and distribution act as microdetectors of the radiation in the same way that
are difficult to study by other methods. they respond to light in photographic film. After an adequate
exposure time in lightproof boxes, the slides are developed
photographically. Silver bromide crystals reduced by the radia-
Scanning Electron Microscopy tion produce small black grains of metallic silver, which under
Scanning electron microscopy (SEM) provides a high- either the light microscope or TEM indicate the locations of
resolution view of the surfaces of cells, tissues, and organs. Like radiolabeled macromolecules in the tissue (Figure 1–9).
the TEM, this microscope produces and focuses a very narrow Much histological information becomes available by
beam of electrons, but in this instrument the beam does not autoradiography. If a radioactive precursor of DNA (such
pass through the specimen (Figure 1–8b). Instead, the surface as tritium-labeled thymidine) is used, it is possible to know
of the specimen is first dried and spray-coated with a very thin which cells in a tissue (and how many) are replicating DNA
FIGURE 1–9â•‡ Microscopic autoradiography.
G
G
a b
Autoradiographs are tissue preparations in which particles called (a) Black grains of silver from the light-sensitive material coating
silver grains indicate the cells or regions of cells in which specific the specimen are visible over cell regions with secretory granules
macromolecules were synthesized just prior to fixation. Shown and the duct indicating glycoprotein locations. (X1500)
here are autoradiographs from the salivary gland of a mouse (b) The same tissue prepared for TEM autoradiography shows sil-
injected with 3H-fucose 8 hours before tissue fixation. Fucose was ver grains with a coiled or amorphous appearance again localized
incorporated into oligosaccharides, and the free 3H-fucose was mainly over the granules (G) and in the gland lumen (L). (X7500)
removed during fixation and sectioning of the gland. Autoradio- (Figure 1–9b, used with permission from Drs Ticiano G. Lima and
graphic processing and microscopy reveal locations of newly syn- A. Antonio Haddad, School of Medicine, Ribeirão Preto, Brazil.)
thesized glycoproteins containing that sugar.
and preparing to divide. Dynamic events may also be analyzed.

For example, if one wishes to know where in the cell protein is
›â•ºENZYME HISTOCHEMISTRY
produced, if it is secreted, and its path in the cell before being Enzyme histochemistry (or cytochemistry) is a method for
secreted, several animals are injected with a radioactive amino localizing cellular structures using a specific enzymatic activ-
acid and tissues collected at different times after the injections. ity present in those structures. To preserve the endogenous
Autoradiography of the tissues from the sequential times will enzymes histochemical procedures usually use unfixed or
indicate the migration of the radioactive proteins. mildly fixed tissue, which is sectioned on a cryostat to avoid
adverse effects of heat and organic solvents on enzymatic
›â•ºCELL & TISSUE CULTURE activity. For enzyme histochemistry (1) tissue sections are
immersed in a solution containing the substrate of the enzyme
Live cells and tissues can be maintained and studied outside to be localized; (2) the enzyme is allowed to act on its sub-
the body in culture (in vitro). In the organism (in vivo), cells strate; (3) the section is then put in contact with a marker
are bathed in fluid derived from blood plasma and containing compound that reacts with a product of the enzymatic action
many different molecules required for survival and growth. on the substrate; and (4) the final product from the marker,
Cell culture allows the direct observation of cellular behavior which must be insoluble and visible by light or electron
under a phase-contrast microscope and many experiments microscopy, precipitates over the site of the enzymes, identify-
technically impossible to perform in the intact animal can be ing their location.
accomplished in vitro. Examples of enzymes that can be detected histochemi-
The cells and tissues are grown in complex solutions of cally include the following:
known composition (salts, amino acids, vitamins) to which ■âŒ€ Phosphatases, which remove phosphate groups from
serum or specific growth factors are added. Cells to be cultured macromolecules (Figure 1–10).
are dispersed mechanically or enzymatically from a tissue or ■âŒ€ Dehydrogenases, which transfer hydrogen ions from
organ and placed with sterile procedures in a clear dish to one substrate to another, such as many enzymes of the
which they adhere, usually as a single layer (Figure 1–5). Such citric acid (Krebs) cycle, allowing histochemical identifi-
preparations are called primary cell cultures. Some cells can cation of such enzymes in mitochondria.
be maintained in vitro for long periods because they become ■âŒ€ Peroxidase, which promotes the oxidation of sub-
immortalized and constitute a permanent cell line. Most cells strates with the transfer of hydrogen ions to hydrogen
obtained from normal tissues have a finite, genetically pro- peroxide.
grammed life span. However certain changes (some related
to oncogenes; see Chapter 3) can promote cell immortality, a
process called transformation, and are similar to the initial › â•ºâ•º MEDICAL APPLICATION
changes in a normal cell’s becoming a cancer cell. Improve-
Many enzyme histochemical procedures are used in the
ments in culture technology and use of specific growth factors
medical laboratory, including Perls’ Prussian blue reaction for
now allow most cell types to be maintained in vitro.
iron (used to diagnose the iron storage diseases, hemochro-
As shown in Chapter 2, incubation of living cells in vitro
matosis and hemosiderosis), the PAS-amylase and alcian blue
with a variety of new fluorescent compounds that are seques-
reactions for polysaccharides (to detect glycogenosis and
tered and metabolized in specific compartments of the cell
mucopolysaccharidosis), and reactions for lipids and sphin-
provides a new approach to understanding these compart-
golipids (to detect sphingolipidosis).
ments both structurally and physiologically. Other histologic
techniques applied to cultured cells have been particularly
important for understanding the locations and functions of
microtubules, microfilaments, and other components of the
cytoskeleton. ›â•ºVISUALIZING SPECIFIC MOLECULES
A specific macromolecule present in a tissue section may also
be identified by using tagged compounds or macromolecules
› â•ºâ•º MEDICAL APPLICATION that bind specifically with the molecule of interest. The com-
Cell culture is very widely used to study molecular changes pounds that interact with the molecule must be visible with
that occur in cancer; to analyze infectious viruses, myco- the light or electron microscope, often by being tagged with a
plasma, and some protozoa; and for many routine genetic or detectible label. The most commonly used labels are fluores-
chromosomal analyses. Cervical cancer cells from a patient cent compounds, radioactive atoms that can be detected with
later identified as Henrietta Lacks, who died from the disease autoradiography, molecules of peroxidase or other enzymes
in 1951, were used to establish one of the first cell lines, that can be detected with histochemistry, and metal (usually
called HeLa cells, which are still used in research on cellular gold) particles that can be seen with light and electron micros-
structure and function throughout the world. copy. These methods can be used to detect and localize specific
sugars, proteins, and nucleic acids.
Visualizing Specific Molecules 11
Examples of molecules that interact specifically with

FIGURE 1–10â•‡ Enzyme histochemistry.
other molecules include the following:
C H A P T E R
■âŒ€ Phalloidin, a compound extracted from mushroom,
Amanita phalloides, interacts strongly with the actin pro-
tein of microfilaments.
L ■âŒ€ Protein A, purified from Staphylococcus aureus bacte-
ria, binds to the Fc region of antibody molecules, and
can therefore be used to localize naturally occurring or
1
applied antibodies bound to cell structures.
Histology & Its Methods of Study â•‡ ■â•‡ Visualizing Specific Molecules
■âŒ€ Lectins, glycoproteins derived mainly from plant seeds,
bind to carbohydrates with high affinity and specificity.
Different lectins bind to specific sugars or sequences of
sugar residues, allowing fluorescently labeled lectins to
be used to stain specific glycoproteins or other macro-
molecules bearing specific sequences of sugar residues.
Immunohistochemistry
A highly specific interaction between macromolecules is that
between an antigen and its antibody. For this reason labeled
antibodies are routinely used in immunohistochemistry
L L
to identify and localize many specific proteins, not just
those with enzymatic activity that can be demonstrated by
histochemistry.
aa The body’s immune cells interact with and produce anti-
bodies against other macromolecules—called antigens—that
are recognized as “foreign,” not a normal part of the organism,
and potentially dangerous. Antibodies belong to the immu-
noglobulin family of glycoproteins and are secreted by lym-
phocytes. These molecules normally bind specifically to their
Ly provoking antigens and help eliminate them.
Widely applied for both research and diagnostic pur-
poses, every immunohistochemical technique requires an
antibody against the protein that is to be detected. This means
Ly that the protein must have been previously purified using bio-
chemical or molecular methods so that antibodies against it
can be produced. To produce antibodies against protein x of a
certain animal species (eg, a human or rat), the isolated pro-
tein is injected into an animal of another species (eg, a rabbit
or a goat). If the protein’s amino acid sequence is sufficiently
b N different for this animal to recognize it as foreign—that is, as
an antigen—the animal will produce antibodies against the
protein.
(a) Micrograph of cross sections of kidney tubules treated Different groups (clones) of lymphocytes in the injected
histochemically to demonstrate alkaline phosphatases (with animal recognize different parts of protein x and each clone
maximum activity at an alkaline pH) showing strong activity of produces an antibody against that part. These antibodies are
this enzyme at the apical surfaces of the cells at the lumens (L)
of the tubules. (X200)
collected from the animal’s plasma and constitute a mixture
of polyclonal antibodies, each capable of binding a different
(b) TEM image of a kidney cell in which acid phosphatase has
been localized histochemically in three lysosomes (Ly) near the region of protein x.
nucleus (N). The dark material within these structures is lead It is also possible, however, to inject protein x into a
phosphate that precipitated in places with acid phosphatase mouse and a few days later isolate the activated lymphocytes
activity. (X25,000) and place them into culture. Growth and activity of these cells
(Figure 1–10b, used with permission from Dr Eduardo
can be prolonged indefinitely by fusing them with lymphocytic
Katchburian, Department of Morphology, Federal University of
São Paulo, Brazil.) tumor cells to produce hybridoma cells. Different hybridoma
clones produce different antibodies against the several parts
of protein x and each clone can be isolated and cultured sepa- The indirect method is used more widely in research and
rately so that the different antibodies against protein x can be pathologic tests because it is more sensitive, with the extra
collected separately. Each of these antibodies is a monoclo- level of antibody binding serving to amplify the visible signal.
nal antibody. An advantage to using a monoclonal antibody Moreover, the same preparation of labeled secondary antibody
rather than polyclonal antibodies is that it can be selected to can be used in studies with different primary antibodies (spe-
be highly specific and to bind strongly to the protein to be cific for different antigens) as long as all these are made in the
detected, with less nonspecific binding to other proteins that same species. There are other indirect methods that involve the
are similar to the one of interest. use of other intermediate molecules, such as the biotin-avidin
In immunohistochemistry a tissue section that one technique, which are also used to amplify detection signals.
believes contains the protein of interest is incubated in a solu- Examples of indirect immunocytochemistry are shown in
tion containing antibody (either monoclonal or polyclonal) Figure 1–12, demonstrating the use of this method with cells
against this protein. The antibody binds specifically to the in culture or after tissue sectioning for both light microscopy
protein and after a rinse the protein’s location in the tissue or and TEM.
cells can be seen with either the light or electron microscope
by visualizing the antibody. Antibodies are commonly tagged › â•ºâ•º MEDICAL APPLICATION
with fluorescent compounds, with peroxidase or alkaline
Because cells in some diseases, including many cancer cells,
phosphatase for histochemical detection, or with electron-
often produce proteins unique to their pathologic condition,
dense gold particles for TEM.
immunohistochemistry can be used by pathologists to diag-
As Figure 1–11 indicates, there are direct and indirect
nose many diseases, including certain types of tumors and
methods of immunocytochemistry. The direct method just
some virus-infected cells. Table 1-1 shows some applications
involves a labeled antibody that binds the protein of interest.
of immunocytochemistry routinely used in clinical practice.
Indirect immunohistochemistry involves sequential
application of two antibodies and additional washing steps. The
(primary) antibody specifically binding the protein of interest Hybridization Techniques
is not labeled. The detectible tag is conjugated to a second- Hybridization usually implies the specific binding between
ary antibody made in an animal species different (“foreign”) two single strands of nucleic acid, which occurs under appro-
from that which made the primary antibody. For example, pri- priate conditions if the strands are complementary. The greater
mary antibodies made by mouse lymphocytes (such as most the similarities of their nucleotide sequences, the more read-
monoclonal antibodies) are specifically recognized and bound ily the complementary strands form “hybrid” double-strand
by antibodies made in a rabbit or goat injected with mouse molecules. Hybridization at stringent conditions allows the
antibody immunoglobulin. specific identification of sequences in genes or RNA. This can
FIGURE 1–11â•‡ Immunocytochemistry techniques.
Labeled
secondary
Labeled Unlabeled antibody
antibody primary
antibody
Antigen Antigen
Tissue section
Glass slide
Direct Indirect
Immunocytochemistry (or immunohistochemistry) can be direct labeled secondary antibody is obtained that was (1) made in
or indirect. Direct immunocytochemistry (left) uses an antibody another species against immunoglobulin proteins (antibodies)
made against the tissue protein of interest and tagged directly from the species in which the primary antibodies were made and
with a label such as a fluorescent compound or peroxidase. When (2) labeled with a fluorescent compound or peroxidase. When
placed with the tissue section on a slide, these labeled antibod- the labeled secondary antibody is applied to the tissue section, it
ies bind specifically to the protein (antigen) against which they specifically binds the primary antibodies, indirectly labeling the
were produced and can be visualized by the appropriate method. protein of interest on the slide. Because more than one labeled
Indirect immunocytochemistry (right) uses first a primary secondary antibody can bind each primary antibody molecule,
antibody made against the protein (antigen) of interest and labeling of the protein of interest is amplified by the indirect
applied to the tissue section to bind its specific antigen. Then a method.
Visualizing Specific Molecules 13
FIGURE 1–12â•‡ Cells and tissues stained by immunohistochemistry.
1 C H A P T E R
Histology & Its Methods of Study â•‡ ■â•‡ Visualizing Specific Molecules
a c
throughout the cytoplasm. Primary antibodies against the

filament protein desmin and fluorescein isothiocyanate
(FITC)–labeled secondary antibodies were used in the indirect
staining technique, with the nucleus counterstained blue with
DAPI. (X650)
(b) A section of small intestine treated with an antibody against
the enzyme lysozyme. The secondary antibody labeled with
peroxidase was then applied and the localized brown color
produced histochemically with the peroxidase substrate
3,3′-diamino-azobenzidine (DAB). The method demonstrates
lysozyme-containing structures in scattered macrophages and in
the large clusters of cells. Nuclei were counterstained with hema-
toxylin. (X100)
(c) A section of pancreatic cells in a TEM preparation incubated
with an antibody against the enzyme amylase and then with
protein A coupled with gold particles. Protein A has high affin-
ity toward antibody molecules and the resulting image reveals
b
the presence of amylase with the gold particles localized as very
small black dots over dense secretory granules and developing
granules (left). With specificity for immunoglobulin molecules,
Immunocytochemical methods to localize specific proteins can labeled protein A can be used to localize any primary antibody.
be applied to either light microscopic or TEM preparations using a (X5000)
variety of labels. (Figure 1–12c, used with permission from Dr Moise Bendayan,
(a) A single cultured uterine cell stained fluorescently Departments of Pathology and Cell Biology, University of Montreal,
to reveal a meshwork of intermediate filaments (green) Montreal, Canada.)
â•‡
TABLE 1-1 Examples of specific antigens with diagnostic importance.
Antigens Diagnosis
Specific cytokeratins Tumors of epithelial origin

Protein and polypeptide hormones Certain endocrine tumors
Carcinoembryonic antigen (CEA) Glandular tumors, mainly of the digestive tract and breast
Steroid hormone receptors Breast duct cell tumors
Antigens produced by viruses Specific virus infections
occur with cellular DNA or RNA when nucleic acid sequences › â•ºâ•º MEDICAL APPLICATION
in solution are applied directly to prepared cells and tissue sec-
Warts on the skin of the genitals and elsewhere are due to
tions, a procedure called in situ hybridization (ISH).
infection with the human papilloma virus (HPV) which causes
This technique is ideal for (1) determining if a cell has
the characteristic benign proliferative growth. As shown in
a specific sequence of DNA, such as a gene or part of a gene
Figure 1–12 such virus-infected cells can often be demon-
(Figure 1–13), (2) identifying the cells containing specific
strated by ISH. Certain cancer cells with unique or elevated
messenger RNAs (mRNAs) (in which the corresponding
expression of specific genes are also localized in tumors and
gene is being transcribed), or (3) determining the localiza-
studied microscopically by ISH.
tion of a gene in a specific chromosome. DNA and RNA of
the cells must be initially denatured by heat or other agents
to become completely single-stranded and the nucleotide
sequences of interest are detected with probes consisting of
single-stranded complementary DNA (cDNA). The probe
›â•ºINTERPRETATION OF STRUCTURES
IN TISSUE SECTIONS
may be obtained by cloning, by polymerase chain reaction
(PCR) amplification of the target sequence, or by chemical In studying and interpreting stained tissue sections, it is
synthesis if the desired sequence is short. The probe is tagged important to remember that microscopic preparations are the
with nucleotides containing a radioactive isotope (localized by end result of a series of processes that began with collecting
autoradiography) or modified with a small compound such as the tissue and ended with mounting a coverslip on the slide.
digoxigenin (identified by immunocytochemistry). A solution Certain steps in this procedure may distort the tissues slightly,
containing the probe is placed over the specimen under con- producing minor structural abnormalities called artifacts not
ditions allowing hybridization and after the excess unbound present in the living tissue.
probe is washed off, the localization of the hybridized probe is One such distortion is minor shrinkage of cells or tissue
revealed through its label. regions produced by the fixative, by the ethanol, or by the heat
needed for paraffin embedding. Shrinkage can create artificial
spaces between cells and other tissue components. Such spaces
FIGURE 1–13â•‡ In situ hybridization (ISH). can also result from the loss of lipids or low-molecular-weight
substances not preserved by the fixative or removed by the
dehydrating and clearing fluids. Slight cracks in sections may
also appear as large spaces in the tissue.
Other artifacts may include small wrinkles in the section
(which the novice may confuse with linear structures in tissue)
and precipitates from the stain (which may be confused with
cellular structures such as cytoplasmic granules). Students must
be aware of the existence of artifacts and able to recognize them.
Another difficulty in the study of histologic sections is the
impossibility of differentially staining all tissue components on
one slide. A single stain can seldom demonstrate well nuclei,
mitochondria, lysosomes, basement membranes, elastic fibers,
etc. With the light microscope, it is necessary to examine prepa-
rations stained by different methods before an idea of the whole
composition and structure of a cell or tissue can be obtained. The
TEM allows the observation of cells with all its internal struc-
tures and surrounding ECM components, but only a few cells in
a tissue can be conveniently studied in these very small samples.
Finally, when a structure’s three-dimensional volume is
cut into very thin sections, the sections appear microscopically
to have only two dimensions: length and width. When examin-
In situ hybridization of this tissue section with probes for the ing a section under the microscope, the viewer must always keep
human papilloma virus (HPV) reveals the presence of many
cells containing the virus. The section was incubated with a
in mind that components are missing in front of and behind
solution containing a digoxigenin-labeled complementary what is being seen because many tissue structures are thicker
DNA (cDNA) probe for the HPV DNA. The probe was then than the section. Round structures seen microscopically may
visualized by direct immunohistochemistry using peroxidase- actually be portions of spheres or tubes. Because structures in
labeled antibodies against digoxigenin. This procedure stains a tissue have different orientations, their two-dimensional (2D)
brown only those cells containing HPV. (X400; H&E)
(Used with permission from Dr Jose E. Levi, Virology Lab,
appearance will also vary depending on the plane of section. A
Institute of Tropical Medicine, University of São Paulo, Brazil.) single convoluted tube will appear in a tissue section as many
separate rounded or oval structures (Figure 1–14).
Interpretation of Structures in Tissue Sections 15
FIGURE 1–14â•‡ Interpretation of 3D structures in 2D sections.
1 C H A P T E R
Histology & Its Methods of Study â•‡ ■â•‡ Interpretation of Structures in Tissue Sections
In thin sections 3D structures appear to have only two dimensions.
Such images must be interpreted correctly to understand the actual
structure of tissue and organ components. For example, blood ves-
sels and other tubular structures appear in sections as round or oval
shapes whose size and shape depend on the transverse or oblique
angle of the cut. A highly coiled tube will appear as several round
and oval structures. In TEM sections of cells, round structures may
represent spherical organelles or transverse cuts through tubular
organelles such as mitochondria. It is important to develop such
interpretive skill to understand tissue and cell morphology in micro-
scopic preparations.
Histology & Its Methods of Studyâ•‡ SUMMARY OF KEY POINTS
Preparation of Tissues for Study Autoradiography

■âŒ€ Chemical fixatives such as formalin are used to preserve tissue ■âŒ€ This process localizes cell components synthesized using radioactive
structure by cross-linking and denaturing proteins, inactivating precursors by detecting silver grains produced by weakly emitted
enzymes, and preventing cell autolysis or self-digestion. radiation in a photographic emulsion coating the tissue section or cells.
■âŒ€ Dehydration of the fixed tissue in alcohol and clearing in organic ■âŒ€ With either light microscopy or TEM, autoradiography permits
solvents prepare it for embedding and sectioning. unique studies of processes such as tissue growth (using radioactive
■âŒ€ Embedding in paraffin wax or epoxy resin allows the tissue to be DNA precursors) or cellular pathways of macromolecular synthesis.
cut into very thin sections (slices) with a microtome.
■âŒ€ Sections are mounted on glass slides for staining, which is required to Cell & Tissue Culture
reveal specific cellular and tissue components with the microscope. ■âŒ€ Cells can be grown in vitro from newly explanted tissues (primary
■âŒ€ The most commonly used staining method is a combination of the cultures) or as long-established cell lines and can be examined in the
stains hematoxylin and eosin (H&E), which act as basic and acidic living state by phase-contrast light microscopy.
dyes, respectively. Enzyme Histochemistry
■âŒ€ Cell substances with a net negative (anionic) charge, such as DNA
and RNA, react strongly with hematoxylin and basic stains; such ■âŒ€ Histochemical (or cytochemical) techniques use specific enzy-
material is said to be “basophilic.” matic activities in lightly fixed or unfixed tissue sections to produce
■âŒ€ Cationic substances, such as collagen and many cytoplasmic proteins visible products in the specific enzyme locations.
react with eosin and other acidic stains and are said to be “acidophilic.” ■âŒ€ Fixation and paraffin embedding denatures most enzymes, so histo-
chemistry usually uses frozen tissue sectioned with a cryostat.
Light Microscopy ■âŒ€ Enzyme classes for which histochemical study is useful include
■âŒ€ Bright-field microscopy, the method most commonly used by phosphatases, dehydrogenases, and peroxidases, with peroxidase
both students and pathologists, uses ordinary light and the colors often conjugated to antibodies used in immunohistochemistry.
are imparted by tissue staining. Visualizing Specific Molecules
■âŒ€ Fluorescence microscopy uses ultraviolet light, under which only
fluorescent molecules are visible, allowing localization of fluores- ■âŒ€ Some substances specifically bind certain targets in cells.
cent probes which can be much more specific than routine stains. ■âŒ€ Immunohistochemistry is based on specific reactions between an
■âŒ€ Phase-contrast microscopy uses the differences in refractive antigen and antibodies labeled with visible markers, often fluores-
index of various natural cell and tissue components to produce an cent compounds or peroxidase for light microscopy and gold par-
image without staining, allowing observation of living cells. ticles for TEM.
■âŒ€ Confocal microscopy involves scanning the specimen at succes- ■âŒ€ If the cell or tissue antigen of interest is detected by directly binding
sive focal planes with a focused light beam, often from a laser, and a labeled primary antibody specific for that antigen, the process is
produces a 3D reconstruction from the images. considered direct immunohistochemistry.
■âŒ€ Indirect immunohistochemistry uses an unlabeled primary anti- Interpretation of Structures in Tissue Sections
body that is detected bound to its antigen with labeled secondary ■âŒ€ Many steps in tissue processing, slide preparation, and staining can
antibodies. introduce minor artifacts such as spaces and precipitates that are
■âŒ€ The indirect immunohistochemical method is more commonly not normally present in the living tissue and must be recognized.
used because the added level of antibody binding amplifies the sig- ■âŒ€ Sections of cells or tissues are essentially 2D planes through 3D
nal detected and provides greater technical flexibility. structures, and understanding this fact is important for their cor-
■âŒ€ Specific gene sequences or mRNAs of cells can be detected micro- rect interpretation and study.
scopically using labeled complementary DNA (cDNA) probes in a
technique called in situ hybridization (ISH).
Histology & Its Methods of Studyâ•‡ ASSESS YOUR KNOWLEDGE
1. In preparing tissue for routine light microscopic study, which 7. Microscopic autoradiography uses radioactivity and can be employed
procedure immediately precedes clearing the specimen with an to study what features in a tissue section?
organic solvent? a. The types of enzymes found in various cell locations
a. Dehydration b. Cellular sites where various macromolecules are synthesized
b. Fixation c. The sequences of mRNA made in the cells
c. Staining d. The dimensions of structures within the cells
d. Clearing e. The locations of genes transcribed for specific mRNA
e. Embedding
8. To identify and localize a specific protein within cells or the extracel-
2. Which of the following staining procedures relies on the cationic lular matrix one would best use what approach?
and anionic properties of the material to be stained? a. Autoradiography
a. Enzyme histochemistry b. Enzyme histochemistry
b. Periodic acid-Schiff reaction c. Immunohistochemistry
c. Hematoxylin & eosin staining d. Transmission electron microscopy
d. Immunohistochemistry e. Polarizing microscopy
e. Metal impregnation techniques
9. In situ hybridization is a histological technique used to visualize what
3. In a light microscope used for histology, resolution and magnifica- type of macromolecule?
tion of cells are largely dependent on which component? a. Proteins
a. Condenser b. Carbohydrates
b. Objective lens c. Certain enzymes
c. Eyepieces or ocular lenses d. Nucleic acids
d. Specimen slide e. Lipids
e. The control for illumination intensity
10. Hospital laboratories frequently use unfixed, frozen tissue specimens
4. Cellular storage deposits of glycogen, a free polysaccharide, could sectioned with a cryostat for rapid staining, microscopic examina-
best be detected histologically using what procedure? tion, and diagnosis of pathological conditions. Besides saving much
a. Autoradiography time by avoiding fixation and procedures required for paraffin
b. Electron microscopy embedding, frozen sections retain and allow study of what macro-
c. Enzyme histochemistry molecules normally lost in the paraffin procedure?
d. Hematoxylin & eosin staining a. Carbohydrates
e. Periodic acid-Schiff reaction b. Small mRNA
c. Basic proteins
5. Adding heavy metal compounds to the fixative and ultrathin sec- d. Acidic proteins
tioning of the embedded tissue with a glass knife are techniques e. Lipids
used for which histological procedure?
a. Scanning electron microscopy
b. Fluorescent microscopy
c. Enzyme histochemistry
d. Confocal microscopy
e. Transmission electron microscopy
6. Resolution in electron microscopy greatly exceeds that of light
microscopy due to which of the following?
a. The wavelength of the electrons in the microscope beam is
shorter than that of a beam of light.
b. The lenses of an electron microscope are of greatly improved
quality.
c. For electron microscopy the tissue specimen does not require
staining.
d. The electron microscope allows much greater magnification of
a projected image than a light microscope provides.
e. An electron microscope can be much more finely controlled
than a light microscope.
Answers: 1a, 2c, 3b, 4e, 5e, 6a, 7b, 8c, 9d, 10e
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sous les étoiles. Quand ainsi la Voix la faisait veuve, elle restait
fidèle cependant et, sans répondre aux cris d’appel de ses frères
sauvages, attendait le retour de Kazan. Parfois, lorsque Kazan
prêtait l’oreille à la Voix, elle le mordillait légèrement, pour lui
témoigner son déplaisir, et grognait vers la Voix.
Or, ce jour-là, comme la Voix retentissait pour la troisième fois,
Louve Grise, au lieu de s’attacher à Kazan et de tenter de le retenir,
lui tourna soudain le dos et se tapit au fond de sa tanière.
Elle s’y coucha, et Kazan ne vit plus, dans l’obscurité, que ses
deux yeux qui flamboyaient, farouches.
Il grimpa vers la pointe extrême du Sun Rock, par la piste étroite,
usée sous ses griffes, et demeura indécis. Depuis la veille, un
certain trouble, qu’il ne pouvait s’expliquer, était en lui. Il y avait du
nouveau qui rôdait sur le Sun Rock. Il ne le voyait point, mais le
sentait.
Il redescendit vers Louve Grise et passa sa tête à l’entrée de la
tanière. Il fut accueilli, non par la plainte caressante coutumière,
mais par un grondement menaçant, qui fit retrousser sur ses crocs
les lèvres de la louve.
Pour la quatrième fois, la Voix se fit entendre et Louve Grise
claqua farouchement des mâchoires. Kazan, après avoir encore
hésité, se décida à descendre du Sun Rock et fila dans la direction
de la cabane.
Par l’instinct de défiance du Wild, qui était en lui, il ne prévenait
jamais de son arrivée, par aucun aboi. Il apparut si soudainement, et
sans crier gare, que Jeanne, qui tenait son bébé dans ses bras,
sursauta, en apercevant dans la porte la tête broussailleuse et les
larges épaules de Kazan. Mais, nullement effrayé, l’enfant s’agita et
se contorsionna de plaisir, et tendit vers le chien-loup ses deux
menottes, en émettant à son adresse un gentil baragouin.
— Kazan ! s’écria Jeanne doucement, avec un geste de
bienvenue. Viens ici, Kazan !
La lueur sauvage, ardente comme du feu, qui luisait dans les
prunelles de Kazan, tomba. Il s’était arrêté, une patte sur le seuil de
la cabane, et semblait répugner à avancer. Puis, tout à coup,
abaissant le panache de sa queue, il s’écrasa sur le sol et entra, en
rampant sur son ventre, comme un chien pris en faute.
Il affectionnait les créatures qui vivaient dans la cabane. Mais la
cabane elle-même, il la haïssait. Car toute cabane sentait le gourdin,
le fouet et la servitude. Pour lui-même, et comme tous les chiens de
traîneau, il préférait, pour dormir, aux parois d’une niche bien close,
le sol couvert de neige et le toit du ciel ou des sapins.
Sous la caresse de la main de Jeanne, Kazan éprouva ce
délicieux petit frisson coutumier, qui était sa récompense, lorsqu’il
quittait pour la cabane Louve Grise et le Wild. Lentement il leva la
tête, jusqu’à ce que son museau noir vînt se poser sur les genoux de
la jeune femme. Puis il ferma béatement les yeux, tandis que la
mystérieuse petite créature qu’était bébé Jeanne l’asticotait, à coups
de pieds mignons, et, de ses mains menues, lui tirait ses poils rudes.
Plus encore que la caresse de la jeune femme, ces jeux enfantins
dont il était l’objet faisaient son bonheur.
Immobile et concentré sur lui-même, comme un sphinx, et
comme pétrifié, Kazan demeurait là sans bouger, respirant à peine.
Le mari de Jeanne n’aimait point à le voir ainsi et s’inquiétait
toujours, en ce cas, de ce que pouvait ruminer le cerveau mystérieux
du chien-loup. Mais la jeune femme était plus confiante en Kazan et
le savait incapable d’une trahison.
— Bon vieux Kazan, disait-elle, je te remercie d’être venu à mon
appel. Bébé et moi nous serons seuls cette nuit. Le papa du petit est
parti au Poste le plus proche et nous comptons sur toi pour nous
garder.
Elle lui chatouillait le museau, du bout de la longue tresse
soyeuse de ses cheveux. Ce qui, quel que fût son désir de n’en point
broncher, faisait souffler et éternuer Kazan, à la grande joie du
marmot.
Puis Jeanne, s’étant levée, s’occupa, durant le reste de la
journée, à empaqueter toutes sortes d’objets qui se trouvaient dans
la cabane, Kazan, qui l’observait, fut fort étonné de ce manège
énigmatique. Quelque chose qu’il pressentait, mais ne pouvait
comprendre, se préparait sans nul doute.
Le soir venu, après l’avoir beaucoup caressé, Jeanne lui dit :
— N’est-ce pas, Kazan, que tu nous défendrais cette nuit, si un
danger nous menaçait ? Maintenant je vais verrouiller la porte, car tu
dois demeurer avec nous jusqu’à demain matin.
Elle se remit à caresser le chien-loup, avec émotion. Sa main
tremblait nerveusement.
— Bientôt, sais-tu, reprit-elle, nous allons partir, nous en
retourner chez nous. Notre récolte de fourrures est terminée. Oui,
nous repartons tout là-bas, là où vivent les parents de mon mari, là
où il y a des villes, avec de grandes églises, des théâtres et des
concerts de musique, et un tas d’autres des plus belles choses qui
sont en ce monde. Et nous t’emmènerons avec nous, Kazan !
Kazan ne comprenait pas ce que lui disait Jeanne, mais il agitait
sa queue, tout heureux de voir que la jeune femme lui parlait. Il en
oubliait Louve Grise et sa rancune contre la cabane, et il alla se
coucher tranquillement dans un coin.
Mais, lorsque Jeanne et l’enfant se furent endormis, quand le
silence fut retombé dans la nuit, son malaise le reprit. Il se mit sur
ses pattes et tourna furtivement tout autour de la chambre, reniflant
les murs, la porte, et tous les objets empaquetés par Jeanne.
Il gémit. La jeune femme, à demi réveillée, l’entendit et murmura :
— Tiens-toi tranquille, Kazan ! Va dormir… Va dormir…
Alors il ne bougea plus et demeura immobile au milieu de la
chambre, inquiet et écoutant.
Et, à travers les murs de bûches de la cabane, il entendit une
plainte lointaine qui venait faiblement jusqu’à lui. C’était le cri de
Louve Grise. Ce cri différait toutefois de celui qu’il avait coutume
d’ouïr. Ce n’était pas un appel de solitude. C’était tout autre chose.
Il courut vivement vers la porte close et se reprit à gémir. Mais
Jeanne et l’enfant dormaient profondément et ne l’entendirent point.
Une fois encore le cri retentit et tout se tut à nouveau. Kazan
s’étala devant la porte et y passa le reste de la nuit.
C’est là que Jeanne le trouva, tout alerté, lorsque le lendemain
matin, de bonne heure, elle s’éveilla. Elle entr’ouvrit la porte et, en
une seconde, il fut dehors. A toute volée, il s’élança vers le Sun
Rock, dont le soleil levant teintait le faîte d’une lueur d’or.
Il y grimpa rapidement, par la piste étroite et raboteuse, et ne
trouva point que Louve Grise fût venue au-devant de lui.
Il atteignit la tanière et huma l’air, le dos hérissé et les pattes
raides. Quelque chose de nouveau flottait dans l’air et ce quelque
chose était de la vie. Il se glissa dans la fente pratiquée entre deux
rochers et, avançant la tête, se trouva nez à nez avec Louve Grise,
qui n’était pas seule.
Louve Grise poussa un gémissement plaintif. Les poils
s’aplatirent sur le dos de Kazan et il répondit par un grognement
attendri. Puis lentement il recula et, dans la lumière de l’aurore, il se
coucha devant l’entrée de la tanière, faisant à la louve, de son corps,
comme un bouclier.
Louve Grise était mère.
XI
LA TRAGÉDIE SUR LE SUN ROCK
Toute cette journée, Kazan demeura sur le Sun Rock. Le

sentiment de sa paternité nouvelle était plus fort que l’appel de la
cabane.
Au crépuscule, Louve Grise se releva de sa couche et tous deux
allèrent faire ensemble un tour sous les sapins. La louve, avec de
petits grognements, mordillait gentiment le cou hirsute de Kazan. Et
Kazan, selon le vieil instinct de ses pères, répondait en caressant de
sa langue la gueule de Louve Grise. Celle-ci témoigna de sa
satisfaction par une série de halètements saccadés, qui étaient sa
manière de rire.
Puis, soudain, elle perçut un cri menu et plaintif qui venait jusqu’à
elle. Elle se hâta de quitter Kazan et de regrimper vers la tanière, où
l’un de ses trois petits l’appelait.
Kazan comprit que Louve Grise ne devait plus quitter, à cette
heure, le sommet du Sun Rock et qu’il devait, seul, se livrer à la
chasse, afin de pourvoir à la nourriture de sa compagne et à celle de
sa couvée. Dès que la lune fut levée, il se mit donc en quête d’un
gibier et, vers l’aube, il revint au gîte, en apportant dans sa mâchoire
un gros lapin blanc. Louve Grise s’en reput gloutonnement. Et Kazan
sut, dès lors, que chaque nuit il devait désormais agir de même.
Le jour suivant, ni celui d’après, il n’alla point vers la cabane, quoi
qu’il entendît la voix de Jeanne et celle de son mari, qui l’appelaient.
Le quatrième jour seulement, il reparut et Jeanne lui fit grande fête,
ainsi que le poupon qui, en babillant, lui envoya force coups de
pieds, qu’il sentait à peine au travers de son épaisse fourrure.
— J’espère, dit le mari de Jeanne, que nous ne nous repentirons
pas un jour de l’avoir emmené avec nous et que son instinct
sauvage ne reprendra jamais le dessus. Mais je me demande
comment tu t’habitueras, vieux diable à notre existence de là-bas.
Cela va te changer fort des forêts où tu as toujours vécu.
— Je ne les regretterai pas moins, mes forêts, répondit Jeanne.
Avec mon pauvre papa, j’y ai, moi aussi, si longtemps vécu ! C’est
pour cela, sans doute, que j’aime tant mon bon vieux Kazan. Après
toi et le bébé, c’est lui, je crois, que j’affectionne le plus sur la terre.
Quant à Kazan, plus encore qu’il ne l’avait déjà éprouvé, il sentit
qu’un événement inconnu se préparait dans la cabane. Ce n’était,
sur le sol, que paquets et ballots.
Toute la semaine, il en fut de même. Kazan en était à ce point
agité que le mari de Jeanne ne put s’empêcher de remarquer, un
soir :
— Je crois qu’il sait réellement. Il a compris que nous préparions
notre départ. Mais quand pourrons-nous partir ? Le fleuve a
recommencé hier à déborder et il est, pour l’instant, impossible d’y
naviguer. Si l’inondation continue, nous serons encore ici dans huit
jours, peut-être plus longtemps.
Quelques jours s’écoulèrent encore et le moment arriva où,
durant la nuit, la pleine lune éclaira de son éblouissante clarté le
sommet du Sun Rock. Louve Grise en profita pour effectuer, avec
ses trois petits, tout titubants derrière elle, sa première sortie hors de
la tanière.
Les trois petites boules velues tombaient et trébuchaient à
chaque pas, et se tassaient contre elle, aussi empotées de leurs
mouvements que le bébé de la cabane. Kazan les observait avec
curiosité. Il les entendait émettre les mêmes sons inarticulés et doux,
les mêmes pleurnichements, et il les regardait comme faisait l’enfant
de Jeanne sur ses deux petites jambes, s’en aller tout de travers sur
leurs quatre pattes molles. Spectacle qui le remplissait d’une joie
infinie.
Lorsque la lune fut au zénith et que la nuit fut presque devenue
l’égale du jour, Kazan s’arracha à la contemplation de sa progéniture
et, dégringolant du rocher, partit en chasse.
Il rencontra, dès l’abord, un gros lapin blanc, qui se sauva entre
ses pattes. Durant un demi-mille, il le poursuivit sans pouvoir le
rejoindre. Il comprit qu’il était plus sage d’abandonner la partie. Il
aurait, en effet, lassé à la course un renne ou un caribou, qui vont
devant eux en ouvrant une large piste, facile à suivre. Il n’en est pas
de même pour le petit gibier, qui se glisse sous les taillis et parmi les
fourrés, et qu’un simple renard est plus apte à atteindre qu’un loup.
Il continua donc à battre la forêt, en furetant silencieusement, à
pas de velours, et il eut la chance de tomber à l’improviste sur un
autre lapin blanc, qui ne l’avait pas entendu. Un bond rapide, puis un
second mirent dans sa gueule le souper escompté de Louve Grise.
Kazan s’en revint en trottinant, tout tranquillement, déposant de
temps à autre sur le soi, pour se reposer les mâchoires, le lapin
botté de neige [19] qui pesait bien sept livres.
[19] Surnom donné couramment, au Canada, aux
lapins blancs.
Parvenu au pied du Sun Rock et à la piste étroite qui s’élevait

vers le sommet, il s’arrêta net. Il y avait sur cette piste l’odeur, tiède
encore, de pas étrangers.
Du coup, le lapin lui en tomba des mâchoires. Une étincelle
électrique courut soudain sur chaque poil de son corps. L’odeur qu’il
flairait n’était point celle d’un lapin, d’une martre ou d’un porc-épic.
C’étaient des griffes acérées qui avaient marqué leur empreinte sur
le sol et escaladé le rocher.
En ce moment lui parvinrent des bruits effrayants et confus, qui
lui firent grimper, d’un seul trait, avec un hurlement terrible, l’abrupt
sentier.
Un peu au-dessous du sommet, dans la blanche clarté lunaire,
sur une étroite plate-forme pratiquée dans le roc et qui surplombait
le vide, Louve Grise était engagée dans une lutte à mort avec un
énorme lynx gris. Elle était tombée sous son adversaire, et poussait
des cris aigus et désespérés.
Après être, un instant, demeuré comme cloué sur place, Kazan,
tel un trait de flèche, se rua à la bataille. Ce fut l’assaut muet et
rapide du loup, combiné avec la stratégie plus savante du husky.
Un autre animal aurait succombé dès la première attaque. Mais
le lynx est la créature la plus souple et la plus alerte de Wilderness.
Aussi les Indiens l’appellent-ils « le Rapide ». Kazan avait visé la
veine jugulaire et avait compté que ses crocs longs d’un pouce, s’y
agripperaient profondément. Le lynx, en une fraction infinitésimale
de seconde, s’était rejeté en arrière et les crocs de Kazan ne
saisirent que la masse cotonneuse et touffue des poils de son cou.
L’adversaire avec lequel il avait à lutter, et qui avait abandonné
Louve Grise, était autrement redoutable qu’un loup ou un husky. Une
fois déjà, il s’était trouvé aux prises avec un lynx, tombé dans une
trappe, et il avait tiré du combat des leçons utiles.
Il savait qu’il ne convient pas de s’efforcer à renverser le lynx sur
le dos, comme on doit le faire avec un autre adversaire. Car le gros
chat du Wild se bat des griffes plus encore que des crocs. Et ces
griffes, coupantes comme autant de rasoirs, ont vite fait alors de
lacérer le ventre de son ennemi et de lui ouvrir les entrailles.
Kazan, derrière lui, entendait Louve Grise, grièvement blessée,
gémir et hurler lamentablement. Il tenta de renouveler son emprise
mortelle à la gorge. Mais, cette fois encore, le coup rata et le lynx
échappa à la mort, de moins d’un pouce. Kazan, pourtant, l’avait
solidement saisi et il ne le lâcha plus.
Les deux bêtes demeurèrent étroitement aux prises. Les griffes
du gros chat labouraient les côtes de Kazan, sans atteindre,
heureusement, aucune partie vitale. Soudain, si exigu était l’espace
où ils combattaient, les deux adversaires arrivèrent, sans qu’ils s’en
rendissent compte, au bord du rocher et, brusquement, culbutèrent
ensemble dans le vide.
Ce fut une chute de cinquante à soixante pieds, durant laquelle
Kazan et le lynx tournèrent plusieurs fois sur eux-mêmes. Ils luttaient
avec une telle rage que les crocs de Kazan ne perdirent point leur
emprise et que le lynx continuait à jouer des pattes et des griffes.
Le heurt de leurs deux corps contre le sol fut si rude qu’il les
sépara et les envoya rouler à une douzaine de pieds l’un de l’autre.
Kazan s’était aussitôt relevé et avait couru vers le lynx afin de
reprendre la bataille. Mais le lynx gisait par terre, immobile et
flasque, inondé du sang qui, à gros bouillons, ruisselait de sa gorge.
Ce que voyant, prudemment il s’approcha, reniflant, et sur la
défensive contre toute ruse éventuelle. Il put constater pourtant que
la victoire lui restait et que le gros félin était bien mort. Alors il se
traîna vers Louve Grise.
Il la retrouva dans la tanière, près des corps de ses trois petits,
que le lynx avait mis en pièces. Elle pleurnichait et se lamentait en
face d’eux. Kazan, pour la consoler, se mit à lui lécher la tête et les
épaules, qui saignaient. Durant tout le reste de la nuit, elle continua
à gémir affreusement.
Ce fut seulement lorsque le jour fut venu qu’il fut loisible à Kazan
de constater, en son entier, le terrible travail du lynx. Louve Grise
était aveugle. Ses yeux étincelants étaient désormais fermés à la
lumière, non pas pour un jour ou pour une nuit, mais pour toujours.
D’obscures ténèbres, qu’aucun soleil ne pourrait plus percer, avaient
mis leur linceul sur ses prunelles.
Et, par cet instinct naturel qui est dans la bête pour tout ce qu’elle
ne saurait raisonner, Kazan comprit que sa compagne était, de ce
moment, devenue plus impuissante dans la vie que les infortunées
petites créatures qui, quelques heures auparavant, gambadait
autour d’elle.
En vain, toute la journée, Jeanne l’appela. Lorsque sa voix
arrivait au Sun Rock, Louve Grise, qui l’entendait, se pressait
davantage, apeurée, contre Kazan. Kazan rabattait incontinent ses
oreilles et plus affectueusement la léchait.
Vers la fin de l’après-midi, Kazan descendit du Sun Rock et,
après avoir battu quelque temps la forêt, en rapporta à Louve Grise
un lapin blanc. Elle passa son museau sur le poil de la bête, en flaira
la chair, mais refusa de manger.
Désolé, Kazan songea que le voisinage des louveteaux morts
causait son chagrin et, en la poussant, avec de petits aboiements
engageants, il l’invita à descendre du tragique rocher. Louve Grise le
suivit et, tout en glissant, en atteignit la base. Puis, sans cesser de
toucher du museau le flanc de son guide, elle partit avec lui, sous les
sapins.
Le premier fossé un peu profond qu’il fallut franchir l’arrêta et de
son impuissance, plus encore, Kazan se rendit compte. Vainement il
l’invitait à s’élancer et à sauter avec lui. Elle pleurnichait, et vingt fois
se coucha sur le sol, avant d’oser le bond nécessaire. Elle le risqua
enfin, d’un saut raide et sans souplesse, et s’abattit lourdement près
de Kazan. Et moins que jamais, après cela, elle s’éloigna de lui, si
peu que ce fût. Elle sentait que pour être en sûreté, son flanc ne
devait plus quitter le flanc de son compagnon, ni son museau son
épaule.
Ils firent ainsi un demi-mille. Louve Grise, qui apprenait à
marcher dans sa cécité, chancelait et tombait à tout moment. Un
lapin chaussé de neige étant apparu dans le crépuscule, Kazan
courut à sa poursuite et, instinctivement, après avoir effectué une
vingtaine de bonds, regarda en arrière si Louve Grise le suivait. Elle
était demeurée immobile, comme figée sur ses pattes. Il abandonna
le lapin et revint vers elle.
Ensemble ils passèrent la nuit dans un fourré et, le lendemain
seulement, Kazan, y laissant Louve Grise, alla rendre visite à la
cabane.
Il y trouva la jeune femme et son mari, qui s’aperçurent aussitôt
des blessures, mal cicatrisées, qu’il portait aux épaules et aux
flancs.
— Ce ne peut être qu’avec un ours ou un lynx qu’il s’est battu,
remarqua l’homme. Un loup ne lui aurait point fait de pareilles
blessures.
Ces blessures, Jeanne les pansa de sa main douce. Tout en
parlant à Kazan, elle les lava à l’eau tiède, les oignit d’une salutaire
pommade, et Kazan en éprouva un bien-être délicieux. Une demi-
heure durant, il se reposa sur le pan de la robe de la jeune femme,
qui s’était assise tout exprès, près de lui. Puis il la regarda
béatement, qui allait et venait dans la chambre, rangeait des
paquets mystérieux et préparait le repas.
Le jour s’écoula ainsi, loin de la forêt oubliée, et la lune était déjà
levée lorsque Kazan se décida à rejoindre Louve Grise. Si Jeanne
avait pu deviner que la pauvre bête, pour qui Kazan désormais était
tout dans la vie, le soleil, les étoiles, la lune et la subsistance, gisait
dans son fourré, elle aurait été elle-même secourir Louve Grise.
Mais elle l’ignorait.
Huit jours passèrent encore, durant lesquels, reprenant ses
vieilles habitudes, Kazan partagea son temps entre le fourré et la
cabane. Puis, une après-midi, l’homme lui mit au cou un collier muni
d’une solide lanière, qu’il attacha ensuite à un crampon de fer, dans
le mur de bûches.
On l’y laissa, toute la journée et toute la nuit, et le lendemain, dès
le point du jour, Jeanne et son mari furent debout.
Le mari de Jeanne prit dans ses bras le bébé et sortit le premier.
Jeanne suivit, tenant en main la lanière et Kazan. La porte de la
cabane fut refermée et solidement verrouillée, des planches et des
troncs de jeunes arbres furent cloués extérieurement sur les volets,
et le cortège descendit vers la berge du fleuve, où une grande
pirogue attendait, toute chargée. La veille, un autre homme était
venu et avait emmené avec lui le traîneau et les chiens de l’attelage.
Dans la pirogue Jeanne monta d’abord, avec l’enfant et avec
Kazan, qu’elle fit coucher auprès d’elle. Elle prit place au gouvernail,
son mari se saisit des rames et l’embarcation s’éloigna du rivage, au
fil de l’eau.
La jeune femme, tenant toujours en main la lanière qui retenait
Kazan, se retourna vers la cabane, qui allait disparaître derrière les
arbres, et la salua d’un geste. Une grande émotion était empreinte
sur son visage. Ses yeux étaient humides de larmes.
— Adieu ! Adieu ! s’écria-t-elle.
— Tu n’as pas de chagrin au moins ? lui demanda son mari. Cela
te contrarie que nous partions ?
— Non, non ! répondit-elle vivement. Mais j’aimais ces belles
forêts et cette sauvage nature. Presque toute ma vie s’y est écoulée,
en compagnie de mon pauvre père, dont je laisse le corps derrière
moi. Aucune ville, pour moi, ne vaudra jamais cela.
Un sanglot s’étrangla dans sa gorge et elle reporta ses yeux vers
le bébé…
Depuis quelques minutes, la pirogue, prise par le courant, filait
rapidement.
Se détachant d’une des rives du large fleuve, une longue bande
de sable s’avançait dans l’eau et formait une sorte de presqu’île
plate et dénudée.
Le mari de Jeanne, l’appelant, lui montra, sur cette langue
sablonneuse, une petite tache sombre qui allait et venait. Elle
reconnut Louve Grise, Louve Grise dont les yeux éteints se
tournaient vers Kazan. A défaut de la vue, l’air, qu’elle humait, lui
disait que Kazan, et l’homme et la femme avec lui, s’en allaient, s’en
allaient, s’en allaient…
Kazan s’était dressé, raide, sur ses pattes, et regardait.
Louve Grise, cependant, qui comprenait, au bruit des rames, que
la pirogue s’éloignait, était venue tout au bord de l’eau. Là, s’étant
assise sur son derrière, elle leva la tête vers ce soleil qui pour elle
n’avait plus de rayons, et jeta à l’adresse de Kazan une longue et
retentissante clameur.
La lanière glissa de la main de Jeanne. La pirogue fit une
formidable embardée et un gros corps brun troua l’air. Kazan avait
disparu.
Déjà l’homme avait pris et épaulé son fusil dans la direction de
Kazan, qui nageait.
— Le gredin ! dit-il. Il nous brûle la politesse ! Je vais
t’apprendre…
Non moins rapide, Jeanne avait arrêté son geste. Elle était toute
pâle et cria :
— Non ! ne tire pas ! Laisse-le retourner vers elle ! Laisse-le,
laisse-le !… Là est sa place !
Kazan, ayant atteint le rivage, secoua ses poils ruisselants. Une
dernière fois, il regarda vers la pirogue qui emmenait Jeanne et qui,
quelques minutes après, avait disparu.
Louve Grise l’avait emporté !
XII
DANS LES JOURS DU FEU
De plus en plus, désormais, Kazan oubliait son ancienne vie de

chien de traîneau. Ce n’était plus pour lui qu’une lointaine
réminiscence, comme ces souvenirs effacés qui remontent parfois
en nous, semblables à des feux dans la nuit.
La naissance et la mort des louveteaux, la tragédie terrible du
Sun Rock, le combat avec le lynx et la cécité de Louve Grise, qui en
avait résulté, puis le départ de Jeanne et du bébé, occupaient seuls
son esprit.
La vengeance tirée du lynx n’avait pas rendu la vue à Louve
Grise et c’était pour Kazan un désappointement perpétuel qu’elle ne
fût plus capable de chasser avec lui, dans la plaine infinie ou dans la
forêt obscure. Aussi sa rancune contre les tribus de lynx était-elle
vivace et profonde, et il était devenu l’ennemi mortel de toute la race.
Non seulement il attribuait au lynx la cécité de Louve Grise et la
mort des louveteaux, mais encore le départ de Jeanne et de l’enfant.
Et, chaque fois que son flair découvrait l’odeur du gros chat gris, il
devenait furieux comme un démon, grimaçant et grognant en
retroussant ses lèvres sur ses longs crocs. Toute l’ancestrale
sauvagerie du Wild reparaissait en lui.
Un nouveau code de vie s’était établi peu à peu entre Kazan et
sa compagne aveugle. Lorsqu’ils cheminaient ensemble, Louve
Grise avait appris à ne point le perdre, en se tenant flanc à flanc,
épaule à épaule avec lui, et Kazan, de son côté, savait, poux qu’ils
demeurassent unis, qu’il ne devait point bondir, mais toujours trotter.
Il comprenait aussi qu’il devait choisir un terrain facilement
accessible aux pattes de Louve Grise. Et, s’il arrivait à un endroit
qu’il fallait franchir d’un bond, il touchait Louve Grise de son museau
et poussait de petits cris plaintifs. Alors elle dressait les oreilles et
prenait son élan. Mais, comme elle ne pouvait calculer la longueur
exacte du saut nécessaire, elle sautait toujours, afin de ne point
risquer de tomber à mi-route, plus loin qu’il n’était utile. Ce qui,
parfois, présentait aussi ses inconvénients. Ainsi, les deux animaux
en étaient arrivés à se comprendre.
Enfin, l’odorat et l’ouïe s’étaient, en compensation de la vue
perdue, développés avec plus d’acuité chez Louve Grise. Et toujours
Kazan, qui l’avait remarqué, observait sa compagne et s’en référait à
elle, s’il s’agissait, soit d’écouter un bruit suspect, soit de humer l’air
ou de flairer une piste.
Au moment où la pirogue avait disparu, un instinct plus infaillible
que le raisonnement avait dit à Kazan que Jeanne, son bébé et son
mari étaient partis pour ne plus revenir. Et cependant, de la tanière
où il s’était installé pour l’été, avec Louve-Grise, sous un épais
bouquet de sapins et de baumiers, proche du fleuve, il s’obstina,
chaque jour, des semaines durant, à venir interroger la cabane.
Impatient, il guettait quelque signe de vie. Mais la porte ne
s’ouvrait jamais. Les planches et les petits troncs d’arbres étaient
toujours cloués aux volets des fenêtres et, de la cheminée, pas une
spirale de fumée ne s’élevait. Les herbes et les plantes grimpantes
commençaient à recouvrir le sentier et les murs de bûches, et
l’odeur de l’homme, imprégnée à ces murs, qu’il reniflait, se faisait
de plus en plus faible.
Un jour il trouva, sous une des fenêtres closes, un petit mocassin
d’enfant. Il était vieux et usé, noirci par la neige et la pluie. Il suffit
pourtant à faire le bonheur de Kazan, qui se coucha tout à côté et
demeura là de longues heures. Et, durant ce temps, à ce même
moment, le bébé, à des milliers de milles de distance, était en train
de se divertir avec les jouets merveilleux inventés par la civilisation.
Ce ne fut qu’à la fin de la journée que Kazan s’en alla rejoindre
Louve Grise, parmi les sapins et les baumiers.
Il n’y avait que dans ces visites à la cabane que Louve Grise
n’accompagnait pas Kazan. Tout le reste du temps, les deux bêtes
étaient inséparables. Lorsqu’elles avaient pisté un gibier, Kazan
prenait la chasse, et Louve Grise l’attendait. Les lapins blancs
étaient leur pâture ordinaire. Par une belle nuit de clair de lune, il
arriva, une fois, à Kazan de fatiguer à la course un jeune daim et de
le tuer. Comme la proie était trop lourde pour qu’il pût la rapporter à
Louve Grise, il courut la chercher et la ramena vers le lieu du festin.
Puis advint le grand incendie.
Louve Grise en saisit l’odeur alors que le feu était encore à deux
jours à l’ouest. Le soleil, ce soir-là, se coucha dans un nuage blafard
et sinistre. La lune, qui lui succéda, à l’opposé du ciel, parut toute
rouge et pourprée. Lorsqu’elle surgit ainsi du désert, les Indiens la
nomment la « Lune Saignante » et l’air s’emplit pour eux de
présages funestes.
Le lendemain matin, Louve Grise devint étrangement nerveuse
et, vers midi, Kazan, à son tour, flaira dans l’air l’avertissement
qu’elle avait perçu bien des heures avant lui. L’odeur, de minute en
minute, augmentait d’intensité et, un peu plus tard dans la journée,
le soleil se voila d’une couche de fumée.
Le feu, qui courait dans les bois et les forêts de sapins et de
baumiers, avait commencé par faire rage dans la direction du nord.
Puis le vent sauta du sud à l’ouest, rabattant en direction contraire
les colonnes de fumée. Il devenait de plus en plus probable que
l’incendie ne s’arrêterait qu’au bord du fleuve, vers lequel le brasier
mouvant pourchassait devant lui mille bêtes affolées.
Pendant la nuit qui suivit, le ciel continua à s’embraser d’une
immense lueur fuligineuse et, lorsque le jour parut, la chaleur et la
fumée devinrent intenables et suffocantes.
Saisi de panique, Kazan s’évertuait à trouver un moyen
d’échapper. Il lui eût été facile, quant à lui, de traverser le fleuve à la
nage. Mais Louve Grise, qu’il n’avait point quittée une seconde, s’y
refusait. Dès le premier contact de ses griffes avec l’eau, au bord de
laquelle il l’avait amenée, elle s’était reculée, en contractant tous ses
muscles. A douze reprises différentes, il s’élança dans le courant et
nagea en l’appelant. Tout ce à quoi Louve Grise consentit, ce fut à
s’avancer dans l’eau tant qu’elle avait pied. Puis, avec obstination,
elle revenait toujours en arrière.
Maintenant on pouvait entendre le sourd mugissement du feu.
Élans, rennes, daims, caribous se jetaient à l’eau et, fendant le
courant, gagnaient sans peine la rive opposée. Un gros ours noir,
accompagné de ses deux oursons, qui se traînaient lourdement, fit
de même, et les petits le suivirent. Kazan le regarda, de ses yeux
ardents, et se mit à gémir vers Louve Grise, qui se refusait à bouger.
D’autres bêtes sauvages du Wild, qui redoutaient l’eau autant
qu’elle, et ne voulaient pas, ou ne pouvaient pas nager, vinrent se
réfugier sur la bande de sable étroite et dénudée qui, un peu plus
loin, s’avançait dans le fleuve.
Il y avait là un gros et gras porc-épic, une petite martre aux
formes sveltes, et un chat-pêcheur, qui n’arrêtait pas de renifler l’air
et de geindre comme un enfant. Des centaines d’hermines se
pressaient sur le sable clair, pareilles à une légion de rats, et leurs
petites voix perçantes formaient un chœur ininterrompu. De
nombreux renards couraient, affolés, à la recherche d’un arbre
abattu par le vent en travers du fleuve, qui pût leur servir de pont
pour passer sur l’autre rive. Mais le fleuve était trop large. Il y avait
aussi des frères de race de Louve Grise, des loups, qui hésitaient
devant une traversée à la nage.
Ruisselant d’eau et haletant, à demi suffoqué par la chaleur et la
fumée, Kazan vint prendre place au côté de Louve Grise. Il
comprenait que le seul refuge qui leur restât était la langue de sable
et il se mit en devoir d’y conduire sa compagne.
Comme ils approchaient du petit isthme qui reliait le sable à la
rive, ils sentirent leurs narines se crisper, et n’avancèrent plus
qu’avec précaution. Leur flair leur disait qu’un ennemi n’était pas
loin.
Ils ne tardèrent pas, en effet, à découvrir un gros lynx, qui avait
pris possession du passage et qui, couché sur le sol, s’étalait
largement à l’entrée de la bande de sable. Trois porcs-épics, ne
pouvant passer outre, s’étaient mis en boule, leurs piquants alertés
et frémissants. Un chat-pêcheur, se trouvant dans le même cas,
grognait timidement vers le lynx qui, ayant aperçu Kazan et Louve
Grise, rabattit, ses oreilles et commença à se mettre en garde.
Déjà Louve Grise, pleine d’ardeur et oubliant sa cécité, allait
bondir vers l’ennemi. Kazan, avec un grondement irrité, l’arrêta net,
d’un coup d’épaule, et elle demeura sur place, à la fois écumante et
plaintive, tandis que Kazan marchait seul à la bataille.
A pas légers, les oreilles pointées en avant, sans aucune
menace apparente dans son attitude, il s’avança. C’était la
meurtrière stratégie du husky, habile en l’art de tuer.
Un homme coutumier de la civilisation n’eût pas manqué de
penser que le chien-loup s’approchait du lynx avec des intentions
tout amicales. Mais le lynx savait à quoi s’en tenir. L’instinct
ancestral lui avait appris, à lui aussi, qu’il était en face d’un ennemi.
Ce qu’il ignorait toutefois, c’est que la tragédie du Sun Rock avait fait
cet ennemi plus féroce encore.
Le chat-pêcheur avait compris de son côté qu’une grande bataille
allait se livrer, et il s’écrasa contre terre, au ras du sol. Quant aux
porcs-épics, ils piaulaient éperdument, comme de petits enfants qui
ont grand’peur, et ils dressaient, plus droits, leurs piquants.
Parmi les nuages de fumée, de plus en plus denses, le lynx
s’était aplati sur son ventre, comme font les félins, le train de derrière
ramassé et contracté pour l’élan.
Autour de lui, Kazan se mit à tourner, léger et presque
impondérable, et le lynx pivotait sur lui-même, non moins alerte et
rapide. Huit pieds environ les séparaient.
Ce fut le lynx qui, pareil à une boule, bondit le premier sur son
adversaire. Kazan ne tenta point d’échapper en sautant de côté. Il
para de l’épaule et, comme il avait plus de poids, il encaissa le choc
sans broncher. Le gros chat fut projeté en l’air, avec les lames de
rasoir de ses vingt griffes, et retomba lourdement sur le sol, les
membres en marmelade.
Kazan profita de l’avantage du moment et, sans perdre de temps,
s’élança sur la nuque du lynx.
Louve Grise, à son tour, avait bondi. Sous l’arrière-train de
Kazan, elle implanta ses mâchoires dans une des pattes de derrière
de l’ennemi. L’os craqua.
Le lynx, accablé par le double poids qui pesait sur lui, tenta un
sursaut désespéré. Entraînant avec lui Kazan et la louve, qui purent
heureusement se dégager à temps, il alla retomber sur un des
porcs-épics qui se trouvaient là. Une centaine des redoutables
aiguilles lui entrèrent dans le corps. Fou de douleur et hurlant
comme un possédé, il prit la fuite et, se précipitant dans le brasier, y
disparut parmi la fumée.
Kazan se garda de l’y poursuivre. Le chat-pêcheur gisait comme
un mort, épiant Kazan et Louve Grise de ses petits yeux noirs et
féroces. Les porcs-épics piaulaient et jacassaient pire que jamais,
comme pour implorer grâce.
La flamme, cependant, avait atteint le rivage. L’air était brûlant
comme une fournaise. Kazan et Louve Grise se hâtèrent de venir se
mettre à l’abri, à l’extrémité de la langue de sable, que recouvrait
entièrement une nappe de fumée.
Ils s’y roulèrent sur eux-mêmes, en cachant leur tête sous leur
ventre. Le rugissement de l’incendie ressemblait à celui d’une
grande cataracte et l’on entendait d’énormes craquements, qui
étaient ceux des arbres qui s’écroulaient. L’air s’emplissait de
cendres et de brûlantes étincelles. Plusieurs fois, Kazan dut se
dérouler et, se relevant, secouer les brandons enflammés qui
tombaient sur lui, poussés par le vent, et qui lui roussissaient le poil
et brûlaient la peau, comme autant de fers rouges.
Sur les rives du fleuve poussait, le pied dans l’eau, une épaisse
rangée de broussailles vertes, où le feu s’arrêta. Le couple s’y
réfugia. Puis la chaleur et la fumée commencèrent à diminuer
d’intensité.
Mais ce ne fut qu’après un temps assez long que Kazan et Louve
Grise purent dégager leurs têtes et respirer plus librement. Sans
cette propice bande de sable, ils eussent été complètement rôtis.
Car, partout en arrière d’eux, la nature était devenue toute noire et le
sol était entièrement calciné.
La fumée s’éclaircit enfin. Le vent remonta au nord et à l’est, et la
dissipa ou refoula, tout en rafraîchissant l’atmosphère.
Le chat-pêcheur, le premier, se décida à regagner la terre et à
s’en retourner dans ce qui demeurait de la forêt. Mais les porcs-
épics étaient encore enroulés lorsque Kazan et Louve Grise se
décidèrent à quitter leur asile.
Ils marchèrent toute la nuit suivante, en longeant la rive du
fleuve, dont ils remontèrent le courant. La cendre était chaude et leur
brûlait douloureusement les pattes. La lune était rouge encore et
sinistre, et semblait toujours un éclaboussement de sang dans le
ciel.
Durant les longues heures où cheminèrent côte à côte les deux
bêtes, tout était silence autour d’elles. Rien, pas même le hululement
d’une chouette. Car, devant le grand feu, tous les oiseaux avaient fui
aussi, à tire-d’aile, sur l’autre rive. Aucun signe de vie ne subsistait
sur cette terre qui, hier encore, constituait, pour les hôtes sauvages
du Wild, un paradis.
Kazan savait que, pour trouver sa nourriture et celle de Louve
Grise, il lui fallait aller plus loin, beaucoup plus loin.
A l’aurore, le couple arriva à un endroit où le fleuve, déjà moins
large, formait une sorte de marais.
Des castors y avaient construit une digue, grâce à laquelle Kazan
et Louve Grise purent enfin passer sur la rive opposée, où la terre
redevenait verte et féconde.

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Junqueira’s Basic Histology Text and

Atlas Anthony L. Mescher

Junqueira’s Basic Histology Text and Atlas Anthony L.

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1 Histology & Its Methods 5 Connective Tissueâ•‡ 96

2 The Cytoplasmâ•‡ 17 7 Cartilageâ•‡129

Neural Plasticity & Regenerationâ•‡ 187 15 Digestive Tractâ•‡ 295

17 The Respiratory Systemâ•‡ 349

Summary of Key Pointsâ•‡ 411 22 The Female Reproductive Systemâ•‡ 460

H istology is the study of the tissues of the body and

FIGURE 1–1â•‡ Sectioning fixed and embedded tissue.

(a) Fixation Dehydration Clearing Infiltration Embedding

Embedding & Sectioning Staining

FIGURE 1–3â•‡ Components and light path of a Fluorescence Microscopy

Histology & Its Methods of Study â•‡ ■â•‡ Light Microscopy

FIGURE 1–4â•‡ Appearance of cells with fluorescent microscopy.

FIGURE 1–5â•‡ Unstained cells’ appearance in three types of light microscopy.

the specimen allows them to be digitally reconstructed into a

Histology & Its Methods of Study â•‡ ■â•‡ Light Microscopy

Although a very small spot of light originating from one plane

power of the objective lens. Confocal microscopy (Figure 1–6)

FIGURE 1–8â•‡ Electron microscopes.

Electron gun Electron gun

TEM image Lens SEM image

(a) Transmission electron microscope (b) Scanning electron microscope

Histology & Its Methods of Study â•‡ ■â•‡Autoradiography

FIGURE 1–9â•‡ Microscopic autoradiography.

and preparing to divide. Dynamic events may also be analyzed.

Examples of molecules that interact specifically with

FIGURE 1–11â•‡ Immunocytochemistry techniques.

FIGURE 1–12â•‡ Cells and tissues stained by immunohistochemistry.

throughout the cytoplasm. Primary antibodies against the

Specific cytokeratins Tumors of epithelial origin

FIGURE 1–14â•‡ Interpretation of 3D structures in 2D sections.

Histology & Its Methods of Studyâ•‡ SUMMARY OF KEY POINTS

Preparation of Tissues for Study Autoradiography

Histology & Its Methods of Studyâ•‡ ASSESS YOUR KNOWLEDGE

Toute cette journée, Kazan demeura sur le Sun Rock. Le

Parvenu au pied du Sun Rock et à la piste étroite qui s’élevait

De plus en plus, désormais, Kazan oubliait son ancienne vie de

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