Chapter 1

Preparation of Tissues for Study

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■ Chemical fixatives such as formalin are used to preserve tissue

structure by cross-linking and denaturing proteins, inactivating

enzymes, and preventing cell autolysis or self-digestion.

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■ Dehydration of the fixed tissue in alcohol and clearing in organic

solvents prepare it for embedding and sectioning.

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■ Embedding in paraffin wax or epoxy resin allows the tissue to be

cut into very thin sections (slices) with a microtome.

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■ Sections are mounted on glass slides for staining, which is

required to reveal specific cellular and tissue components with the

microscope.

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■ The most commonly used staining method is a combination of the

stains H&E, which act as basic and acidic dyes, respectively.

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■ Cell substances with a net negative (anionic) charge, such as DNA

and RNA, react strongly with hematoxylin and basic stains; such

material is said to be “basophilic.”

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■ Cationic substances, such as collagen and many cytoplasmic pro-

teins react with eosin and other acidic stains and are said to be

“acidophilic.”

Light Microscopy

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■ Bright-field microscopy, the method most commonly used by

both students and pathologists, uses ordinary light and the colors

are imparted by tissue staining.

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■ Fluorescence microscopy uses UV light, under which only fluo-

rescent molecules are visible, allowing localization of fluorescent

probes which can be much more specific than routine stains.

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■ Phase-contrast microscopy uses the differences in refractive

index of various natural cell and tissue components to produce an

image without staining, allowing observation of living cells.

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■ Confocal microscopy involves scanning the specimen at succes-

sive focal planes with a focused light beam, often from a laser, and

produces a 3D reconstruction from the images.

Autoradiography

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■ This process localizes cell components synthesized using radioactive

precursors by detecting silver grains produced by weakly emitted

radiation in a photographic emulsion coating the tissue section or cells.

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■ With either light microscopy or TEM, autoradiography permits

unique studies of processes such as tissue growth (using radioactive

DNA precursors) or cellular pathways of macromolecular synthesis.

Cell & Tissue Culture

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■ Cells can be grown in vitro from newly explanted tissues (primary

cultures) or as long-established cell lines and can be examined in the

living state by phase-contrast light microscopy.

Enzyme Histochemistry

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■ Histochemical (or cytochemical) techniques use specific enzy-

matic activities in lightly fixed or unfixed tissue sections to produce

visible products in the specific enzyme locations.

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■ Fixation and paraffin embedding denatures most enzymes, so histo-

chemistry usually uses frozen tissue sectioned with a cryostat.

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■ Enzyme classes for which histochemical study is useful include

phosphatases, dehydrogenases, and peroxidases, with peroxidase

often conjugated to antibodies used in immunohistochemistry.

Visualizing Specific Molecules

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■ Some substances specifically bind certain targets in cells.

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■ Immunohistochemistry is based on specific reactions between an anti-

gen and antibodies labeled with visible markers, often fluorescent com-

pounds or peroxidase for light microscopy and gold particles for TEM.

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■ If the cell or tissue antigen of interest is detected by directly binding

a labeled primary antibody specific for that antigen, the process is

considered direct immunohistochemistry.

■ Indirect immunohistochemistry uses an unlabeled primary anti-

body that is detected bound to its antigen with labeled secondary antibodies

The indirect immunohistochemical method is more commonly used

because the added level of antibody binding amplifies the signal

detected and provides greater technical flexibility.

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■ Specific gene sequences or mRNAs of cells can be detected micro-

scopically using labeled cDNA probes in a technique called in situ

hybridization (ISH).

Interpretation of Structures in Tissue Sections

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■ Many steps in tissue processing, slide preparation, and staining can

introduce minor artifacts such as spaces and precipitates that are not

normally present in the living tissue and must be recognized.

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■ Sections of cells or tissues are essentially 2D planes through 3D

structures, and understanding this fact is important for their correct

interpretation and study.