STAINING

- Applying dyes to see and study patterns and physical characteristics of cells.
- Done to make the tissue on the slide visible to the light microscope.
- There are two most commonly used kind of staining in Histopathology, namely:
 Acidic, which stains nucleus. It has high affinity to basic dyes.
 Basic, it stains cytoplasm. It has high affinity to acidic dyes.

There are 4 methods of staining:

1. Direct Staining- using aqueous or alcoholic dyes, staining with simple solution of dyes.
2. Indirect Staining- stain- mordant  tissue
3. Progressive Staining- in definitive sequence (no acid alcohol), continued until desired intensity is
achived.
4. Regressive Staining- absolute (over stained) then excess is decolorized (addition of acid alcohol)

Histological Staining – Demo in section by direct interaction with dye (color is active tissue component)

Bacterial, specific tissue stains

Histochemical Staining – Tissues are studied thru chemical reactions that permit localization of specific
tissue substance.

Metachromatic Staining- specific dye, color that is different with that of the stain.

H & E- It is the most common for anatomical studies. Best for surgical specimens.

Hematoxylin- most common (basic, for nucleus) the active coloring is HEMATIN (from oxidation)

Over oxidation- oxyhematein formation (color turns brown). The mordant used is: Alum and Iron

Eosin- (Acidic, cytoplasmic) counterstain after hematoxylin and before methylene blue. It is the most
widely used cytoplasmic stain. It is best staining at pH 4.6-5.0

H&E Staining steps

1. 2x Xylol – Deparaffinization
2. Desending grade of alcohol- Hydration
3. Stain with Harriss Hematoxylin
4. Differentiate with acid alcohol
5. Blue in Ammonia water, Scotts h20
6. Stain with Eosin
7. Ascending grade of alcohol- dehydration
8. Clean 2x with Xylol
9. Mount
10. label