[Histonet] Bielschowsky's stain

From:

"Robin Turcotte"

Does anyone have good result with BIELSCHOWSK'S STAIN ?

I am using the technique from Bancroft as follows on brain tissue of dogs:

1. Deparaffinize and hydrate to distilled water. Wash well in distilled water.

2. Place sections in 50 ml of 20% silver nitrate in the dark at 37 C. Keep this silver solution for

stage 4.

3. Wash well in distilled water.

4. Add strong ammonia to 50 ml of silver nitrate (from stage 2) until the initial precipitate disappears.

Leave section in this solution for 10 minutes at 37 C.

I would to understand step 4. Should I add concentrated ammonia?

I added concentrated ammonia 1 to 2 drop at a time. Immediately I got precipitate that did not disappear.

I repeated this step with dilute ammonia getting the same result. I filtered the soluton and continued the

staining and obtained no color on my axons.

I guess I have a problem with step 4. How much volume of ammonia should be added at what concentration. Is there a critical point in this staining.

Thank you

Robin