Hematoxylin and Eosin staining : principle, procedure and interpretation

Hematoxylin and Eosin (H & E) staining is the most common staining technique in histopathology. This uses a combination of two dyes, Hematoxylin and Eosin used for demonstration of nucleus and cytoplasmic inclusions in clinical specimens.

Principle

Alum acts as mordant and hematoxylin containing alum stains the nucleus light blue. This turns red in presence of acid, as differentiation is achieved by treating the tissue with acid solution. Bluing step converts the initial soluble red color within the nucleus to an insoluble blue color. The counterstaining is done by using eosin which imparts pink color to the cytoplasm.

Reagents

1. Harri’s Hematoxylin stain

A = 1 gm hematoxylin in 10 ml ethanol
B = 20 gm ammonium alum in hot distilled water
Mix A & B, boil and add 0.5 gm of mercuric oxide and filter.

2. Eosin solution

Yellow eosin = 1 gm
Distilled water = 80 ml
Ethanol = 320 ml
Glacial Acetic Acid = 2 drops

3. 0.5% HCl
4. Dilute ammonia water

Procedure

1. Deparaffinize the section : flame the slide on burner and place in the xylene. Repeat the treatment.
2. Hydration : Hydrate the tissue section by passing through decreasing concentration of alcohol baths and water. (100%, 90%, 80%, 70%)
3. Stain in hematoxylin for 3-5 minutes
4. Wash in running tap water until sections “blue” for 5 minutes or less.
5. Differentiate in 1% acid alcohol (1% HCl in 70% alcohol) for 5 minutes.
6. Wash in running tap water until the sections are again blue by dipping in an alkaline solution (eg. ammonia water) followed by tap water wash.
7. Stain in 1% Eosin Y for 10 minutes
8. Wash in tap water for 1-5 minutes
9. Dehydrate in increasing concentration of alcohols and clear in xylene
10. Mount in mounting media
11. Observe under microscope

Result and Interpretation

• Nuclei : blue, black
• Cytoplasm : Pink
• Muscle fibres : deep red
• RBCs : orange red
• Fibrin : deep pink