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Tissue Preparation

Both light and electron microscopy share the basic preparative

methods with slight modifications for the specific type of analysisrequired.

Each specific method has its limitations and associated artifacts,

which must be borne in mind while interpreting histologic

images. Specimen preparation is necessary:

1 to keep tissue from falling apart,

2 to cut sections thin enough so details can be resolved,

3 to stain biological material which is ordinarily transparent.

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Basic Steps of Tissue Preparation

Fixation (chemical preservation of biological structure)

1. Coagulating/precipitation, e.g., picric acid (Bouin's fluid).

2. Chemical crosslinking, e.g., formaldehyde

Dehydration (alcohols) & clearing (xylene)

Embedding (paraffin wax for LM; epoxy resin for EM)

Trimming & sectioning (5-10 m m)

Mounting sections, de-waxing and hydration

Staining & washing

Application of coverslip with mounting media

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Staining

Hematoxylin & Eosin (H&E), most commonly used in your lab:

Hematoxylin: Cationic, positively charged, blue dye complex (with Al3+)

Reacts with negativelycharged groups: e.g., COO- in proteins SO4-- in

proteoglycans (GAGs) PO4-- in nucleic acids

reacts with basophilic structures

Eosin: Anionic, negatively charged, pink dye

Reacts with positively charged groups: e.g., NH3+ in proteins

Reacts with acidophilic structures

Others (chemistry not well understood):

Azan: stains collagen blue-green.

Iron hematoxylin: stains mitochondria. Silver: e.g., stains Golgi apparatus, and reticular fibers (collagen Type III).

Sudan: lipid soluble; stains mitochondria.

Toluidine blue: metachromatic (stacked dye molecule are purple; e.g., stain

mast cells.

Verhoeff: stains elastin protein black.

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Hematoxylin & Eosin Staining

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Iron Hematoxylin Staining

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Silver Staining

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Staining

Histochemistry:

Enzymatic:

Examples: Alkaline phosphatase, acid phosphatase, ATPase, etc. Tissue

section is incubated with an appropriate substrate. Either the product

of the reaction is insoluble and precipitates at the site of the reactions,

or it further reacts with a trapping agent which precipitates.

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Acridine Orange Staining

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Staining

Immunohistochemistry and

immunofluorescence

Principle: A primary

antibody is made which

binds to the antigen in the

tissue section. A secondaryantibody, which is labeled

either with a fluorescent

marker or an enzyme, reacts

with the primary antibody.The results is either a

fluorescent signal or a

colored precipitate at the

site of the antigen.

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Electron Microscopy Preparation

Fixation: glutaraldehyde

Post-fixation step: OsO4

Embedding: epoxy (plastic) resins instead of paraffin wax.

Sectioning: ultrathin sections (50 nm) to allow electrons to

penetrate.

Staining: heavy metal salts - lead citrate and uranyl

acetate.