FIXATION:        10% buffered neutral formalin.

TECHNIQUE:        Paraffin sections cut at 5 μm.

SOLUTIONS:        

1% Ferric Chloride

Ferric Chloride -------------------------------------------------------------------        0.3 gm

Distilled water -------------------------------------------------------------------        30.0 ml

       Prepare fresh.

0.4% Potassium Ferricyanide

Potassium ferricyanide ---------------------------------------------------------                0.04 gm

Distilled water -----------------------------------------------------------------                10.0 ml

       Prepare fresh.

Nuclear Fast Red (Kernechtrot) Solution

       Dissolve 0.1 gm nuclear fast red in 100 ml of 5% solution of aluminum

       sulfate with the aid of heat.  Cool, filter, add a few grains of thymol as

       a preservative.

1. Deparaffinize and hydrate to distilled water.
2. Add 10 ml of 0.4% potassium ferricyanide to 30 ml of 1.0% ferric chloride.  Pour into a glass Coplin jar, place slides into the solution and microwave at power level one (60W) for 1 ½ minutes.  Dip the slides up and down several times and allow them to remain in the warm solution for 30 seconds.
3. Wash in running tap water for 1 minute and rinse in distilled water.
4. Counterstain with nuclear fast red solution for 3 minutes.
5. Rinse with three changes of distilled water.
6. Dehydrate in graded alcohols and clear in xylene, three or four changes.
7. Mount with synthetic resin.

Argentaffin, chromaffin, lipofuscin and melanin ----------------------                blue to dark blue

Nuclei --------------------------------------------------------------        red

This procedure will demonstrate the same pigments that stain with the Fontana-Masson silver method which are argentaffin, chromaffin, melanin, and lipofuscin.  Performing the stain in a microwave oven produces better staining results than doing the stain at room temperature.

Culling , C.F.A.:  Handbook of Histopathological and Histochemical Techniques, 3rd Ed., Butterworths, 1974, p. 386.