Use celloidinized 12 micron paraffin wax sections.
Take to distilled water.
Treat sections with acid formalin for at least 5 minutes.
Wash in three changes of distilled water over 5 minutes.
Place in silver solution for 15 minutes at room temperature.
Drain the slide and place in the reducer that has been heated to 40-45°C, agitate gently and leave for 1 minute. The sections should be yellow-brown in colour.
Rinse in 50% alcohol for 5-10 seconds then in three changes of distilled water.
Examine microscopically, if there is insufficient impregnation repeat the procedure from the acid formalin stage. Reduce the time in the silver solution and the temperature of the reducer to 30°C.
Tone in the gold chloride solution - 2 minutes.
Drain and treat with intensifier - 15 seconds.
Wash in water, fix in 5% sodium thiosulphate - 2 minutes.
Wash again in water, dehydrate, (prolong to remove the celloidin) clear and mount.
Nerve fibres - black.
Background - grey.
The intensifier and toning stages may be omitted in which case the
results would be: axons - dark brown/black, background - golden brown.
Formalin - 25ml
1% nitric acid - 0.2ml
Distilled water - 75ml
Silver nitrate - l5g
Potassium nitrate - l0g
5% glycine - 1ml
Distilled water - 100ml
Pyrogallol (pyrogallic acid) - l0g
1% nitric acid - 2ml
Absolute ethanol - 550ml
Distilled water - 450ml
(allow to stand 24 hours before use)
Gold chloride (sodium tetrachloroaurate) - 1g
Distilled water - 200ml
Glacial acetic acid - 0.2ml
50% ethanol - 100ml
Aniline oil - 2 drops