(M. Fero, courtesy of the Porter lab)
Also see Wei-Ming and Emily's protocol This reference link is broken.
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).
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)
- Xylene 3' (x2)
- 100% EtOH (x2)
- 95% EtOH (x1)
- 80% EtOH (x1)
- H2O (x2)
- Replace the dirty xylene #1, above with fresh xylene.
- Replace the dirty 100% EtOH #1, above with fresh 100% EtOH.
(Note: Unless otherwise specified, slides should be dipped 10 times in each solution)
- Hematoxylin, 2 min (x1)
- Running H2O x 2 min
- 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.
- H2O x1
- Ammonia solution 10 dips (x1) (changes the stain from purple to blue)
- Running H2O 5 min
- 80% EtOH x1
- Eosin 15"
- 95% EtOH x2
- 100% EtOH
- Xylene 3 min x2
- Wipe off the xylene off the back of a slide on a paper towel
- Using a clean glass rod add a drop of permount to the slide
- Cover with a glass slip (use 2 drops of permount for large slips)
- Tilt the slide on edge on a paper towel to remove extra xylene or permount
- If bubbles are present gently squeeze them out by pressing on slip with a pencil eraser
- Place slides on a paper towel to cure overnight