FIXATION:                10% buffered neutral formalin.

TECHNIQUE:                Paraffin sections cut at 5 μm.

SOLUTIONS:

Ammoniacal Silver Solution

To 10 ml of 2% silver nitrate (0.2 gm silver nitrate in 10 ml of distilled water) add 5 ml of 0.8% lithium hydroxide, monohydrate (0.04 gm lithium hydroxide in 5 ml of distilled water).  Then add concentrated ammonium hydroxide, drop by drop with constant shaking, until the precipitate just dissolves.  Make up the solution to 200 ml with distilled water and store in a refrigerator at 3o-6o C.  The solution is stable for at least one month.

0.2% Gold Chloride Solution

Gold chloride, 1% aqueous ---------------------------------------------------------                20.0 ml

Distilled water ----------------------------------------------------------------------                80.0 ml

2% Sodium Thiosulfate Solution

Sodium thiosulfate -------------------------------------------------------------------                2.0 gm

Distilled water ------------------------------------------------------------------------              100.0 ml

Nuclear Fast Red (Kernechtrot) Solution

Dissolve 0.1 gm nuclear fast red in 100.0 ml of 5% solution of aluminum sulfate with the aid of heat.  Cool, filter, add a few grains of thymol as a preservative.

• Use control slide.

1. Deparaffinize and hydrate to distilled water.
2. Place slides in 40 ml of refrigerated cold ammoniacal silver solution in a plastic Coplin jar and microwave at power level 6 (360W) for 40 seconds.  Gently agitate the Coplin jar for about 15 seconds.  Microwave again at power level 6 for 40 seconds.  Gently agitate the Coplin jar for about 15 seconds.  Allow the slides to remain in the hot solution for 2-3 minutes or until the sections appear a medium brown.  Check after 2 minutes.
3. Rinse sections with four changes of distilled water.
4. Gold chloride solution for 30 seconds.
5. Rinse with three changes of distilled water.
6. Sodium thiosulfate solution for 30 seconds.
7. Rinse with four changes of distilled water.
8. Nuclear fast red solution for 3 minutes.
9. Rinse with three changes of distilled water.
10. Dehydrate in graded alcohols.
11. Clear in xylene, three or four changes.
12. Mount with synthetic resin.

Argentaffin cell granules, chromaffin, melanin and other

       Silver reducing substances -----------------------------------                black

Nuclei ---------------------------------------------------------------        pink

This procedure is nonspecific for melanin since chromaffin and argentaffin granules as well as pigments such as lipofuscin and iron are also stained.  To demonstrate that the stained material is melanin the melanin bleach procedure must also be done.

For many years we used the Fontana-Masson method to demonstrate argentaffin and melanin using a hot water bath or microwave oven.  We found that dilute ammoniacal silver works better than the Fontana-Masson silver solution, requires much less silver nitrate, and has greater stability.  The ammoniacal silver solution was originally developed for the demonstration of fungi and has for several years been used for that purpose in the Special Stains Laboratory.

Churukian, C.J. and Schenk, E.A.:  Dilute ammoniacal silver as a substitute for methenamine silver to demonstrate pneumocystis carinii and fungi.  Lab. Med. 17:87-90, 1986.

Brinn, N.T.:  Rapid metallic histological staining using the microwave oven.  J. Histotechnology 6:125-129, 1983.

Masson, P.:  Carcinoids and nerve hyperplasia of the appendicular mucosa.  Amer. J. Path. 4:181-212, 1928.