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.

    1. Eosin solution

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

  1. 0.5% HCl
  2. 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

hematoxylin-and-eosin-staining

Nuclei : blue, black

Cytoplasm : Pink

Muscle fibres : deep red

RBCs : orange red

Fibrin : deep pink

15 Responses

  1. Hai i am lab technician , and also bsc(microbiology) i like your artile from many technician technician gain knowledge

  2. hello, i like this we need more of these. why cant we just dewax in xylene rather than flaming, it gives good results.

  3. Differentiate in 1% acid alcohol (1% HCl in 70% alcohol) for 5 minutes. from my long experience in this field I think 5 minutes it very long time only few seconds it enough to remove excess stain

  4. One major issue…. After staining with eosin, when we put the slide in increasing grade of alcohol, the alcohol removes the eosin… What to do of that…. Kindly mail me or msg me if anyone has solution…
    Mohit402@gmail.com.
    8980236234

Leave a Reply

Your email address will not be published. Required fields are marked *