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Tissue Preparation

Both light and electron microscopy share the basic preparative methods with slight modifications for the specific type of analysis required. 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.

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

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.

Hematoxylin & Eosin Staining

Iron Hematoxylin Staining

Silver Staining

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.

Acridine Orange Staining

Staining
Immunohistochemistry and immunofluorescence Principle: A primary antibody is made which binds to the antigen in the tissue section. A secondary antibody, 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.

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.