University of Alabama at Birmingham

School of Medicine

Virtual Microscopy Histology Manual

Peter G. Anderson, D.V.M., Ph.D.

Professor & Director of Pathology Undergraduate Education
Department of Pathology
University of Alabama at Birmingham

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 University of Alabama at Birmingham
School of Medicine

Virtual Microscopy Histology Manual

Contents
1. Histology ..................................................................................................................................... 4
2. The Cell...................................................................................................................................... 7
3. Epithelial Cells ......................................................................................................................... 12
4 Support Cells and the Extracellular Matrix ............................................................................... 17
5 Contractile Cells......................................................................................................................... 21
6 Nervous Tissue........................................................................................................................... 25
7 Blood Cells................................................................................................................................. 37
8 Immune System ......................................................................................................................... 42
9 Heart and Vessels....................................................................................................................... 51
10 Respiratory System .................................................................................................................. 60
11 Alimentary Tract ...................................................................................................................... 68
12 Liver ......................................................................................................................................... 93
13 Musculoskeletal System......................................................................................................... 101
14 Endocrine System .................................................................................................................. 108
15 Urinary System ...................................................................................................................... 125
16 Male Reproductive System .................................................................................................... 137
17 Female Reproductive System ................................................................................................ 148
18 Skin and Breast ...................................................................................................................... 168
19 Special Senses ........................................................................................................................ 177

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 Acknowledgements

This Virtual Microscopy Histology Laboratory Manual is a derivative work from the
laboratory teaching materials produced over many years by anatomists from the University of
Alabama at Birmingham School of Medicine. The instructors who designed the curriculum,
acquired the teaching slide sets, and developed the original version of this laboratory manual
were: George Hand, Ph.D., Jim Sheetz, Ph.D. and Laura Cotlin, Ph.D.

The virtual microscopy slides described in this manual are primarily scans of original
glass slides used in the University of Alabama at Birmingham School of Medicine Cell Biology
and Histology teaching program. Special thanks to Matthew C. Anderson for scanning the glass
slides. Additional virtual microscopy slide files were kindly contributed by: James L. Fishback,
MD, University of Kansas School of Medicine; Mary Ann Sens, MD, PhD, University of North
Dakota School of Medicine; and Richard M. Conran, M.D., Ph.D., Uniformed Services
University of the Health Sciences.

Every effort has been made to ensure that all images used in this manual are believed to
be in the public domain or are incorporated into our material according to “fair use” guidelines
for derivative works. If any of the images used herein are in fact copyright protected please let
us know immediatley and they will be removed.

Peter G. Anderson, D.V.M., Ph.D.

Professor & Director of Pathology Undergraduate Education
Department of Pathology
University of Alabama at Birmingham
Volker Hall, 213
1670 University Boulevard
Birmingham, Alabama 35294-0019
Phone: 205-934-2414
Email Address: pga@uab.edu
Web page: http://peir.net

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1. Histology

Histology is the study of the microscopic structure of biological material and the ways in which
individual components are structurally and functionally related. It is central to medical science
since it stands at the crossroads between biochemistry, molecular biology and physiology on the
one side, and pathologic processes that cause disease on the other. Although often thought of as
an archaic discipline, practical knowledge of histology is in actuality an integral part of modern
investigative techniques and current medical practice

In this laboratory manual we will focus on the basic structure of human tissues. We will
concentrate on structure-function correlations that are important in the understanding of disease
processes. Thus, we will not attempt to provide a comprehensive review of all structures in the
body; instead we will focus just on the structural relationships that are integral to disease.

Almost all of the tissues we will review are human tissues obtained at autopsy or from surgical
biopsies. As a general rule all fresh tissues are fixed in 10% neutral buffered formalin and are
embedded in paraffin wax before cutting microscopic tissue sections. The embedding process
requires dehydration of the tissues using organic solvents, permeation of the tissues with paraffin
wax, and hardening of the wax for cutting. Tissue sections are then cut at 5 to 7 µm in thickness
and placed on glass slides. The tissues are then rehydrated and stained. This dehydration-wax
embedding - rehydration cycle results in dissolution of any lipid materials within the tissues.
This may lead to alterations in the morphology of tissues. However, if you understand the
process you can overlook these artifacts and still make accurate assessments of the tissue. One
classic “artifact” is the loss of fat from liver tissue obtained from a patient with fatty liver. This
leaves holes in the tissue where the fat globules had been situated before they were dissolved
away. These and other classic artifacts will become second nature to you as you review tissue
sections.

Overview of Tissue Preparation and Staining for Microscopy

1. Obtaining tissues - Human material is obtained at autopsy or from surgical biopsies.

2. Fixation - To preserve the tissue, it is placed immediately in a fixative which acts to

preserve the cell and tissue constituents in as lifelike a manner as possible after death. In
postmortem tissue, considerable autolysis may have occurred prior to fixation. Formalin
(10%) is the fixative most often used by pathologists.

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 3. Dehydration - The fixed tissues must be dehydrated in order to embed them in paraffin
for sectioning. Water is removed from the tissues by passing them through a series of
increasingly concentrated solutions of alcohol.

4. Clearing - Absolute alcohol is not miscible with paraffin. Thus, the alcohol must be
removed from the tissue and replaced with an agent that mixes with molten paraffin. The
most commonly used clearing agent is xylene. The xylene makes the tissues translucent
or “clears” them.

5. Embedding - Following clearing, the tissue is placed in the embedding agent, molten
paraffin, and allowed to steep until the tissue is thoroughly infiltrated by the embedding
medium. The preparation is then cooled, the paraffin solidifies, and the block of tissue
can now be cut with a minimum of distortion. The paraffin infiltrates the interstices of
the tissue and thus provides internal support as well as external support for sectioning.

6. Sectioning - The tissue is now cut into very thin slices, usually 5 to 7 µm, with a
microtome. The sections are then mounted on glass slides and stained.

7. Staining - For morphologic study, it is necessary to create color contrasts in the tissues by
staining. Certain terms are used to distinguish the staining reaction of a cell. The term
basophilic indicates that the structure can be stained with the basic dye hematoxylin. All
nuclei are basophilic. Cytoplasmic elements may be either basophilic, acidophilic or,
neutral. Eosin is the most commonly used acid stain and any acid components that stain
positive with eosin are termed eosinophilic.

8. Other stains used in preparing slides – Most slides for histology and pathology are
stained with Hematoxylin and Eosin (H&E). Additional staining techniques are utilized
to demonstrate specific characteristics of tissues. In any staining process variations in the
tissue and the technical procedure may lead to minor color modifications in individual
slides, but, in general, the reactions are as stated below.

a. Masson’s Trichrome stain (hematoxylin, acid fuchsin, and aniline blue): nuclei
stain black or dark blue; cytoplasm stains red by the acid fuchsin; reticular and
collagen fibers stain blue with aniline blue.

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 b. Gomori’s Trichrome stain: Another version of a trichrome stain that stains nuclei -
red-purple; normal muscle myofibrils - green-blue with distinct A and I bands;
intermyofibrillar muscle membranes – red; and interstitial collagen - green

c. Periodic acid-Schiff’s reagent (PAS). The PAS method stains glycogen, mucin,
connective tissue fibers, and other structures that contain carbohydrates, pink, red, or
maroon. The periodic acid converts adjacent 1, 2 glycol groups to aldehydes and the
basic Fuchsin of Schiff’s reagent stains the aldehydes. Sometimes Hematoxylin is
used as a counter stain giving you a PASH.

c. Silver stain. This special procedure employs silver nitrate to specifically

demonstrate reticular fibers, neurofibrils of neurons and granules in enteroendocrine
cells. These structures are stained black whereas other tissue components may take
on a faint gray background stain without revealing detail.

d. Toluidine blue. Used to demonstrate granules in mast cells. Nuclei are deep blue;
mast cell granules are reddish-purple.

e. Verhoeff-Van Gieson stain (VVG). This method is used for identifying elastic
fibers in tissues such as skin, aorta, etc. The elastic fibers will be stained blue-black
and background will be stained yellow.

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