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.
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable.
Other fixatives are likely to be satisfactory. A section adhesive is recommended.
Optionally, tone with 0.1% gold chloride for 10 seconds.
Rinse well with distilled water.
Fix in sodium thiosulphate 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.
Melanin (unbleached) – black
Melanin (bleached) – unstained
Enterochromaffin – black
Lipofuscin – black
Nuclei – red
Background – grey
If using an elevated temperature, check for adequate reduction at short intervals. It should be complete when the section is light brown.
Do not increase the temperature excessively. Reduction is faster, but less selective and sections may detach.
If the solution is crystal clear or has a distinct smell of ammonia, it may indicate that too much ammonium hydroxide
has been added. If so, add drops of silver nitrate until the faintest opalescence is seen.
Toning is optional, but some microscopists prefer the blacker tones that short tonings give.
Reference 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.