Modified Alizarin Red S for Foetal Specimens

EMS26204

Components:

Alizarin S 0.01% Aqueous Solution
Potassium Hydroxide 1% Aqueous Solution
Potassium Hydroxide 10% Aqueous Solution
Glycerol Potassium Hydroxide

Fixation: 

Two or more weeks in 95% alcohol or Formalin after evisceration.

(evisceration omitted for small embryos).

Staining Procedure:

1. Rinse in water and place in Potassium Hydroxide, 1% for a few hours to a week or
2. more (Formalin fixed specimens require approximately 1 month in Potassium Hydroxide, 10% until bones are clearly viable through the soft tissue. Note: If the tissue becomes too soft, harden for 12-24 hours in solution containing equal parts of 95% alcohol, water and glycerin and then return to the Potassium Hydroxide 1%. The Potassium Hydroxide, 1% may be diluted to 0.5% with distilled water during the last few days of clearing.
3. Wash in running water for 12 hours.
4. Stain in Working Alizarin Red S Solution* for 30-60 minutes. Wash in running water for 30 minutes. (Prepare Working Stain by adding 6-10 drops of Potassium Hydroxide, 1% to 100ml of Alizarin Red S, 0.01%. Prepare fresh.)
5. Decolourise in Glycerol-Potassium Hydroxide Solution (C-204-3) for 1-2 weeks.
6. Mount on a glass frame and dehydrate by passing slowly through the following alcohol-glycerin-water mixtures, in the order given: 1:2:7, 2:2:6, 3:3:4, 4:4:2 and finally 5:5 without water.
7. Seal in the final glycerin-alcohol mixture.

Stain Results:

Bones - Red

Soft Tissue - Transparent and unstained

References:

Dawson, A.B., Stain Techn., 1:123-4, 1926

Richmond, G.W. and Bennett, L., Stain Techn., 13:77-79, 1938.

Clark, G., (ed.), Staining Procedures, 3rd edition, Williams & Wilkins, Baltimore, p.137, c. 1973.