Hematoxylin and Eosin (H&E) Staining

Posted on July 15th, 2016 by UNM CC

(M. Fero, courtesy of the Porter lab)
Revised 2/07
Also see Wei-Ming and Emily's protocol  This reference link is broken.

Reagents
Stains (change after every 3rd use)
Harris hematoxylin: 1X stock. Anatech Lmtd. Cat #842. (616) 964-6450
Gill's Hematoxylin: See recipe.
Eosin: 1x stock. Anatech Cat #837.

Solutions (make fresh each day)
Acid alcohol: 75% EtOH + 1/2500 v/v concentrated HCl (156 mL 95% EtOH + 44 mL H2O + 80 µL HCl = 200 mL)
Ammonia Solution: 0.084% (w/v) NH4OH (200 mL H2O + 0.6 mL 28% w/v NH4OH stock).

Organics
Xylene x2 (Make at least 1 fresh)
100% EtOH (Make at least 1 fresh)
95% EtOH x2 (make both fresh)
80% EtOH x1 (make fresh = 168 mL 95% EtOH + 32 mL H2O)

Deparafinization

  1. Xylene 3' (x2)
  2. 100% EtOH (x2)
  3. 95% EtOH (x1)
  4. 80% EtOH (x1)
  5. H2O (x2)
  6. Replace the dirty xylene #1, above with fresh xylene.
  7. Replace the dirty 100% EtOH #1, above with fresh 100% EtOH.

Staining
(Note: Unless otherwise specified, slides should be dipped 10 times in each solution)

  1. Hematoxylin, 2 min (x1)
  2. Running H2O x 2 min
  3. Acid Alcohol x1 ("Differentiation" lightens the staining, especially outside the nucleus) Skip this differentiation step for progressive staining with the weaker Mayer's Hematoxylin stain.
  4. H2O x1
  5. Ammonia solution 10 dips (x1) (changes the stain from purple to blue)
  6. Running H2O 5 min
  7. 80% EtOH x1
  8. Eosin 15"
  9. 95% EtOH x2
  10. 100% EtOH
  11. Xylene 3 min x2

Mounting

  1. Wipe off the xylene off the back of a slide on a paper towel
  2. Using a clean glass rod add a drop of permount to the slide
  3. Cover with a glass slip (use 2 drops of permount for large slips)
  4. Tilt the slide on edge on a paper towel to remove extra xylene or permount
  5. If bubbles are present gently squeeze them out by pressing on slip with a pencil eraser
  6. Place slides on a paper towel to cure overnight

Tags: Fero-Histology

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