Histopathology Tissue Staining

Fixatives

• Formalin is a fixative of choice for IHC and molecular tests, especially the 10% NBF.
• It prevents alterations during processing and preserves tissue morphology.
• It is cheap and stable.
• Tissue can be stored indefinitely, except for immunohistochem specimens.

Glutaraldehyde

• Similar to formalin but much slower in tissue penetration.
• Used mainly for electron microscopy.
• Can overharden tissue, and fixation is best limited to 2 hrs.

Specimen Preparation

• Criteria for rejecting specimens must be identified.
• Different materials are used for gross examination of specimens.
• Failure to form ribbons during sectioning is due to a dull knife or paraffin that is too hard.
• Holes in the sections are due to improper block positioning or excessive dehydration.

Staining of Tissues

• Staining begins with the complete removal of paraffin for water-soluble stain solutions to act on the tissues.
• Nuclear staining involves the use of basic (cationic or positively charged) dyes, forming dye-salt unions, and is "basophilic".
• Cytoplasmic staining depends on the pH of the solution (acidic-pinkish, basophilic-purplish/bluish).
• Progressive staining is a method where the reaction proceeds "forward" and is stopped once the desired intensity of color is achieved.
• Regressive staining involves overstaining the tissue and then decolorizing until the desired element remains stained.

Harris Hematoxylin

• A commonly used nuclear stain in routine H & E stains.
• Ready-to-use preparations are available.
• Now uses sodium iodate instead of mercuric oxide.

Eosin Counterstain

• Most widely used cytoplasmic stain in routine staining.
• Is the sodium salt of a color acid that stains best at pH 4.6-5.

H & E Manual Progressive Staining Procedure

• Involves a series of steps using xylene, absolute alcohol, Harris Hematoxylin, ammonia, and Eosin.

Automated Staining

• Uses linear strippers to transfer slides from one container to the next, leaving the slide in each container for the same amount of time.

Mounting and Labeling of Slides

• Different types of mounting media are used in histotechnology.
• Different types of cover slips are used.
• Steps in cover slipping include applying mounting media, applying a coverslip, and sealing the edges.
• A slide label includes the patient's name, specimen number, and diagnosis.