Protocol
Masson Fontana
Silver Reduction
Materials
- Silver nitrate, 10% aqu.
- Strong ammonium hydroxide (s.g. 0.880)
- Sodium thiosulphate, 3% aqu.
- Gold chloride, 0.1% aqu.
- Neutral red, 1% aqu.
- Mallory bleach
Material Amount Potassium permanganate, 1% aqu. 47.5 mL Sulfuric acid, 3% aqueous 2.5 mL
Preparation of Ammoniacal Silver
- Place 20 mL of the 10% silver nitrate in a flask and add drops of strong ammonium hydroxide while swirling the solution.A precipitate will form at first, but will redissolve as more ammonia is added.
- Stop when it is almost redissolved and is faintly opalescent, then add 20 mL of distilled water.
Tissue Sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended.
Protocol
- Bring sections to water via xylene and ethanol.
- Optionally, remove melanin with the Mallory bleach.
- Bleach in Oxalic acid for a few minutes.
- Rinse with distilled water.
- Treat with ammoniacal silver, either:
- overnight at room temperature, or
- 45-60 minutes at 37°C, or
- 30 minutes at 56°C
- Rinse well with distilled water.
- Optionally, tone with 0.1% gold chloride for 10 seconds.
- Rinse well with distilled water.
- Fix in sodium thiosulfate for 5 minutes.
- Wash well with running tap water.
- Counterstain with neutral red for 1 minute.
- Rinse with tap water.
- Dehydrate with ethanol, clear with xylene and mount with a resinous medium.
Expected Results
- Melanin (unbleached) – black
- Melanin (bleached) – unstained
- Enterochromaffin – black
- Lipofuscin – black
- Nuclei – red
- Background – grey
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- Bancroft, J. D. and Stevens, A. (1977).
Theory and practice of histological techniques.
Churchill Livingstone, Edinburgh, UK. - Gray, Peter. (1954)
The Microtomist’s Formulary and Guide. p. 596
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
