Prepared by
ROY ELLIS
IMVS Division of Pathology
The Queen Elizabeth Hospital
Woodville Road, Woodville, South Australia 5011
Principle
The melanins are a group of brown-black precipitates whose exact chemical structure is not known. The melanins are bound to proteins and the melanin-protein complex is localised in cells within the so-called melanin granules, found within the cytoplasm. Melanin has the ability to reduce solutions of ammoniacal silver nitrate to metallic silver without the use of an external reducing agent. This method is far from being specific for melanin, since it demonstrates other substances which have similar reducing properties, such as argentaffin cell granules and lipofuschins.
Technical Points
1. Formalin fixation is essential for argentaffin substances, but not critical for melanin.
2. A known positive control section must be used to ensure correct demonstration has been achieved.
3. Step 2 - Check the slide microscopically after 15 mins. Time of the ammoniacal silver impregnation depends upon the tissue component to be demonstrated. At room temperature, melanin will require 12 hrs, argentaffin 24 hrs. At 60°C melanin blackens within 20 minutes, argentaffin requires longer to guarantee maximum demonstration, approximately 40 minutes . Excessive heat over long periods may cause the silver solution to precipitate, give non-specific background deposits, and cause precipitation of silver on connective tissue fibres.
4. Step 7 - Slides should be washed well with distilled water. Red background staining results if the neutral red stain is applied directly from tap water.
5. Ammoniacal silver solutions can be explosive when allowed to dry. Immediately after use neutralise the silver solution with saturated sodium chloride and discard.
Method
1. Bring sections to distilled water
2. Treat with ammoniacal silver solution in a closed jar 15 mins
3. Check microscopically and repeat step 2 if necessary
4. Wash well in distilled water
5. Tone with gold chloride 2 mins
6. Wash well with distilled water
7. Fix in 2% aq sodium thiosulphate 2 mins
8. Wash well with distilled water
9. Counterstain with neutral red stain 1 min
10. Rinse in distilled water
11. Rapidly dehydrate well in absolute alcohol, clear and mount
Results
- melanin..........................................black
- argentaffin cell granules.......................black
- some lipofuscins................................black
- chromaffin.......................................black
- nuclei.............................................red             Â
Reagent Formulae
1. Silver solution – Lillies Diammine variant
Concentrated ammonia is added drop by drop to about 40 mls of 5% aq silver nitrate until the initial precipitate is just dissolved. More silver nitrate is added drop by drop until a faint opalescence persists after thorough shaking. The quicker the solution is prepared, the more effective the silver impregnation, that is , very careful measurements and titration which take a longer time are detrimental to the making of this solution - speed and careful accurate technique is required
The solution will keep for about a week at 4oC, but it is preferable to use a freshly prepared solution.
Ammoniacal silver solutions are aqueous suspensions of silver fulminates which are percussive explosives when dry.
2. Neutral Red Stain – acidified
                         neutral red (CI 50040)           1.0 g
                        distilled water                      100.0 mls
                        gl acetic acid                       1.0 ml
Dissolve the dye in the distilled water. Add the acid. Mix well. Filter into the reagent bottle. Keeps well.
References
Lillie RD, 1965, Histopathologic Technique and Practical Histochemistry, 3rd Ed 1965 The Blakiston Division, McGraw Hill Book Company p240