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MICROWAVE MELANIN BLEACH METHOD
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FIXATION: 10% buffered neutral formalin.
TECHNIQUE: Paraffin sections cut at 5 μm.
SOLUTIONS:
0.5% Potassium Permanganate
Potassium permanganate -------------------------------------------------------- 0.5 gm
Distilled water ------------------------------------------------------------------- 100.0 ml
0.25% Potassium Permanganate
0.5% Potassium permanganate -------------------------------------------------- 22.0 ml
Distilled water ------------------------------------------------------------------ 22.0 ml
1% Oxalic Acid
Oxalic acid ---------------------------------------------------------------------- 1.0 gm
Distilled water ------------------------------------------------------------------- 100.0 ml
Nuclear Fast Red
Dissolve 0.1 gm nuclear fast red in 100 ml of 5% solution of
aluminum sulfate with the aid of heat. Cool, filter, and add a few
grains of thymol as a preservative.
1. Deparaffinize and hydrate to distilled water.
2. Place in 44 ml of 0.25% potassium permanganate solution in a glass Coplin jar and microwave at power level 1 for 3 minutes. Agitate for about 15 seconds and allow to remain in the hot solution (about 65˚ C) for 2 minutes. An alternate method is to treat the slides with potassium permanganate for 30 minutes at room temperature.
3. Rinse in three changes of distilled water.
4. Place in oxalic acid for 2 minutes.
5. Wash in running tap water for 1 minute and rinse in distilled water.
6. Nuclear fast red solution 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.
If the yellow-brown or black granules seen on the H&E stain are no longer visible after the bleaching procedure, but are stained black with the Microwave Ammoniacal Silver Method for silver reducing substances, the pigment may be assumed to be melanin.
We routinely do this method on all requests for melanin along with the Microwave Ammoniacal Silver Method. If a suspected malignant melanoma does not stain with the Microwave Ammoniacal Silver Method, we recommend that Schmorls method for reducing substances, the Giemsa or Lillies Nile Blue A be done. These methods may be useful for demonstrating sparse granules in amelanotic melanomas and certain tumors of the brain that may contain melanin such as Schwannomas.
Luna, L.G.: Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd Ed., New York, McGraw-Hill, 1968, pp. 42, 89.
Image Examples:
Diagnostic Application: