FIXATION:        10% buffered neutral formalin.

TECHNIQUE:        Paraffin sections cut at 5 µm.

SOLUTIONS:

2.5% Ferrous Sulfate

Ferrous sulfate (Fe S04 . 7H20 -------------------------------------------------                1.0 gm

Distilled water ----------------------------------------------------------------                40.0 ml

1% Potassium Ferricyanide - Acetic Acid

Potassium ferricyanide ---------------------------------------------------------                0.5 gm

Acetic acid ---------------------------------------------------------------------                0.5 ml

Distilled water -----------------------------------------------------------------                50.0 ml

0.1% Nuclear Fast Red

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

       aluminum sulfate with the aid of heat.  The solution should be

       heated to its boiling point.  Cool, filter, and add a few grains

       of thymol as a preservative.

1.        Deparaffinize and hydrate to distilled water.

2.        Place slides in 2.5% ferrous sulfate for one hour.

3.        Rinse with six changes of distilled water.

4.        Place in 1% potassium ferricyanide in 1% acetic acid for 30 minutes.

5.        Rinse in four changes of distilled water.

6.        Counterstain with nuclear fast red for 3 minutes.

7.        Rinse in three changes of distilled water.

8.        Dehydrate in graded alcohols.

9.        Clear in xylene, three or four changes.

10.        Mount with synthetic resin.

Melanins of skin, eye and pia, and neuromelanin --------------------                dark green

Background --------------------------------------------------------                faint green

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

This method is specific for melanin because it does not stain any of the lipofuscins. 

Lillie, R.D. and Fullmer, H.M.:  Histopathologic Technic and Practical Histochemistry, 4th Ed., New York, McGraw-Hill, 1976, pp. 526-527.