HEMATOXYLIN & EOSIN (H & E) STAIN PROTOCOL
Technique: Cut 10 - 16 micron (12 Ám) sections in cryostat from snap frozen biopsy. Attach one or more sections to a No.11┌2, 22 mm square coverslip
Reagent alochol - HPLC Fisher A995-4 or histological A962, FLAMMABLE store at room temp. in a flammable cabinet
Eosin Y, disodium salt (Sigma #E-6003, store at room temperature)
Harris Hematoxylin Stain, acidified (Lerner Laboratories #1931382)(R.T.)
Permount - Fisher SP15-100, FLAMMABLE HEALTH HAZARD
Xylenes (Fisher #HC700-1GAL, FLAMMABLE, store R.T. in flammable cabinet)
1. Eosin Y, 1 % aqueous (store at room temperature)
Eosin Y dye 1 g
Deionized water 100 ml
2. Harris Hematoxylin, acidified (store at room temperature)
Filter (Baxter #F2217-150, Grade 363, Qualitative) before use
3. Alcohol 50, 70, 80, 95%
Reagent alcohol 50, 70, 80, 95ml
Deionized water 50, 30, 20, 5ml
1. Immerse sections in the filtered Harris Hematoxylin for 1 minute.
2. Rrinse with tap water.
3. Exchange tap water until the water is clear.
4. Immerse sections in EOSIN stain for 1-2 minutes.
5. Rinse with tap water.
6. Exchange tap water until the water is clear.
7. Dehydrate in ascending alcohol solutions (50%,70%,80%,95%x2, 100%x2).
8. Clear with xylene (2X).
9. Mount coverslip onto a labeled glass slide with Permount.
Nuclei and other basophilic structures are blue. Cytoplasm and acidophilic structures are light to dark red.
1. Thompson, Samuel W. SELECTED HISTOCHEMICAL AND HISTOPATHOLOGICAL METHODS, Charles C. Thomas, Springfield, IL, 1966.
2. Sheehan, D.C. and Hrapchak, B.B.: THEORY AND PRACTICE OF HISTOTECHNOLOGY, 2nd Edition; Battelle Memorial Institute, Columbus, OH, 1987.