HEMATOXYLIN AND EOSIN STAINING

1. Staining Solutions

A. Hematoxylin – 1 (Gills, Lerner, etc.)

B. Eosin-Y (ethanolic)

Stock Solution: Dissolve 1 g Eosin Y in 20 ml dH₂ O. Add 80 ml of 95% EtOH.

Working Solution: 1 part stock solution and 3 parts 80% EtOH. For every 100 ml stain, add 0.5 ml acetic acid. Keep in stoppered bottle. The stain can be recycled and filtered before each use.

2. Staining Procedure

A. Deparaffinize the sections in 2 changes of xylene and take through graded EtOH: 100% (2X), 95% (2X), 70%, and dH₂O (2X).

B. Stain the slides in hematoxylin for 18 min. (depending on strength).

C. Wash off excess stain in running tap water.

D. Dip slides in acid alcohol: 99 ml 70% EtOH + 1 ml 1% HCl.

E. Wash slides in running tap water.

F. Blue in 0.1% Sodium Bicarbonate solution for 30 sec to 1 min.

G. Wash in running tap water

H. Rinse in 95% EtOH, 10 dips

I. Dip slides in Eosin Y 2-3 times (15 sec).

J. Dehydrate in 95% EtOH (2X), 100% EtOH (2X). Be sure last change is clean.

K. Xylene (2X). Be sure last change is clean.

L. Mount.

NOTE: Time for hematoxylin may vary with different tissues. If a different strength of hematoxylin is used, the time will also change. (ie. Lerner-2 would take half the time). It is wise to do a test run when chemicals are changed or haven’t been used for awhile.

Revised 10-6-06.