Jill,  Here is a copy of our procedure.  It is very simple and fairly quick 
when you heat the solution in the microwave. We perform this procedure about 
twice a week, so we keep a stock solution of the Alizarian Red S.  Give us a 
buzz if you have any questions.

We also have plenty of controls if you need one.


Barbara Stancel
USDA, FSIS, Eastern Lab, Pathology
Richard B. Russell Center
950 College Station Road
Athens, Georgia   30605
Phone: 706-546-3556
Fax: 706-546-3589


Name:  Okajima's Stain

Purpose: To stain hemoglobin

Techniques: Cut paraffin sections at 4 to 5 microns.  Use control slide.

Solutions:

     10% Phosphomolybdic Acid - Stock (make fresh)
       Phosphomolybdic acid                 10.0 gm
       Distilled water                     100.0 ml

     7.7% Alizarin Red S - Stock  (shelf life: 1 year)
       Alizarin Red S  (C.I. #58005)         7.7 gm
       Distilled water                     100.0 ml

     Working Stain Solution:	shelf life: 10 hours
       10% Phosphomolybdic acid (stock)     10.0 ml
       7.7% Alizarin red S (stock)          30.0 ml
       Mix just before use.  Discard after use.

     Hematoxylin of choice

Procedure:

1. Deparaffinize and hydrate to water.
2. Place in 10% phosphomolybdic acid solution for 1 minute.
3. Wash in water.
4. Preheat 20 ml of working stain for 10 - 15 seconds in microwave.
   Add slides and stain for 10 minutes.
       (or 1-2 hours in non-heated solution)
5. Wash in water.
6. Counterstain in hematoxylin for 30 seconds - 1 minute (we use Gill's)
   (Follow whatever you normally do after your hematoxylin, but don't
    counterstain with Eosin!)
7. Dehydrate, clear, and coverslip with synthetic mounting medium.


Results:    Hemoglobin..............orange to orange red
            Nuclei..................................blue
            Background.....................reddish brown

References:  Sheehan, 2nd ed., pp. 216-217


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