Bielschowsky's Silver Staining Protocol for Nerve Fibers, Axons, Neurofibrillary Tangles and Senile Plaques

 

NovaUltra Special Stain Kits

 

Principle: The nerve fibers are sensitized with a silver solution. The sections are treated with ammoniacal silver, and then reduced to a visible metallic silver.

 

Fixation: 10% formalin

 

Section: Paraffin sections at 10 um thick.

 

Solutions and Reagents:

 

10% Silver Nitrate Stock Solution:

 

      Silver nitrate ------------------------------------- 5 g

      Distilled water ----------------------------------- 50 ml

 

1% Ammonium Hydroxide Solution:

      Ammonium hydroxide, concentrated ---------- 1 ml

      Distilled water ----------------------------------- 100 ml

 

Developer Stock Solution (make fresh):

 

      37-40% Formaldehyde --------------------------- 20 ml

      Citric acid (trisodium dihydrate, Sigma) ----- 0.5 g

      Nitric acid, concentrated ----------------------- 2 drops

      Distilled water ----------------------------------- 100 ml

 

Developer Working Solution (make fresh immediately before use, discard after use):

      Developer Stock Solution ------------------------ 8 drops

      Ammonium hydroxyide, concentrated --------- 8 drops

      Distilled water ------------------------------------ 50 ml

 

5% Sodium Thiosolfate (HYPO):

 

      Sodium Thiosolfate ------------------------------- 5 g

      Distilled water ------------------------------------ 100 ml

   

Procedure:

 

1.   Deparaffinize sections to distilled water and wash three times.

2.   Place slides in pre-warmed (40 ºC) 10% silver nitrate solution and stain for 15 minutes and sections become light brown color (thicker sections may look dark brown).

3.   Place slides in distilled water and wash for 3 times.

4.   To the silver nitrate solution, add concentrated ammonium hydroxide drop by drop until the precipitate formed is JUST clear. Excess ammonia may cause a precipitate or result in a poor impregnation of the fibers. If ammonia is added too much, add few more drops of 10% silver nitrate solutions to make solution JUST becomes cloudy.

5.   Place slides back in this ammonium silver solution and stain in 40 ºC oven for 30 minutes or until sections become dark brown (may not look very dark brown if the sections are only 10 um thick and may look very dark brown if the sections are 30 um thick).

6.   Place slides DIRECTLY (do not wash slides) in developer working solution for about 1 minute or less (reaction may be very fast, so test a slide, check microscopically to determine exact incubation time).

7.   Dip slides for 1 minute in 1% ammonium hydroxide solution to stop the silver reaction.

8.   Wash slides in 3 changes of distilled water.

9.   Place slides in 5% sodium thiosulfate solution for 5 minutes.

10. Wash in 3 changes of distilled water

11. Dehydrate and clear through 95% ethyl alcohol, absolute alcohol and xylene.

12. Mount with resinous medium.

 

Results:

 

       Axons, neurofibrillary tangles and senile plaques ----------- black

       BaBackground --------------------------------------------------- yellow to brown  

 

Positive Controls:

  

       Brain tissue.

 

Notes:

 

       1) Use glass stainers/containers for all procedure. All the glass containers must be completely cleaned with distilled water.

       2) Developing time is critical so test a few slides and check microscopically to determine exact developing time.