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
  2. 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.

  3. Eosin solution
  4. Yellow eosin = 1 gm
    Distilled water = 80 ml
    Ethanol = 320 ml
    Glacial Acetic Acid = 2 drops

  5. 0.5% HCl
  6. 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
About Dhurba Giri
Dhurba Giri is the author at LaboratoryInfo.com, a scientific blog dedicated for Medical Laboratory Professionals. He's a fresh graduate of Bachelor of Science in Medical Laboratory Technology (B.Sc.MLT) from Pokhara University, Nepal. Connect with him on Facebook !

13 Comments on Hematoxylin and Eosin staining : principle, procedure and interpretation

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

  2. dushyanth bollu // April 3, 2016 at 9:02 am // Reply

    Hai this is dushyanth. This information is good for students and technicians.

  3. johnson ondere // April 9, 2016 at 9:08 am // Reply

    thanks for your teachi ng techniques, am astudend in medical lab tech

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

  5. Thid is very useful piece of info

  6. Dear Dhurba Giri, Please contact me through my mail id europathology@pathologyseries.com

  7. what haematoxylin? Harris or delafield

  8. khalid elfakki abdalla ibrahim // March 23, 2017 at 10:37 am // Reply

    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

  9. Dr. Mohit V. Changanj // April 6, 2017 at 2:54 pm // Reply

    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

  10. HaI I am sivagami this information good

  11. I want to know about staining of nucliec acid

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