HEMATOXYLIN & EOSIN (H & E) STAIN PROTOCOL

Technique: Cut 10 - 16 micron (12 µm) sections in cryostat from snap frozen biopsy. Attach one or more sections to a No.11⁄2, 22 mm square coverslip

Reagents:

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)

Solutions:

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

Staining Procedure:

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.

Results:

Nuclei and other basophilic structures are blue. Cytoplasm and acidophilic structures are light to dark red.

REFERENCES:

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.