Bielschowsky’s silver stain protocol

Paul Polak and Douglas Feinstein, University of Illinois, Chicago. 

Bielschowsky's silver stain is a very useful tool to detect nerve fibers. It can be used to stain axons, neurofibrils and senile plaques in the central nervous system. This method is easy to perform and is routinely used in the study of Alzheimer's disease together with antibody staining. 

Print this protocol.

1. Deparaffinize and hydrate sections to dH₂O.  Wash 3X for 3 min. in dH₂O.
2. Place sections in 50 ml 10% silver nitrate in dark at 37ºC for 30 min. Keep this solution after incubation (for use in step 4).
3. Wash 3X for 3 min in dH₂O.
4. Add concentrated ammonium hydroxide dropwise with stirring to the silver nitrate solution reserved from step 2. Add only enough to dissolve the dark initial precipitate but not more.
5. Incubate sections in this solution for 15 min at 37ºC. Again, save this solution for use in step 7.
6. Wash sections in 0.1% ammonium hydroxide 3X for 2 min at room temperature.
7. Add 350 μl developer solution (0.2 ml 37% formaldehyde, 12 ml dH₂O, 12.5 μl 20% nitric acid and 0.05 g citric acid) to the silver hydroxide solution saved from step 4.
8. Stain sections in this solution for 10 min until they turn black.
9. Wash in 0.1% ammonium hydroxide 3X for 2 min and dH₂O 3X for 2 min.
10. Tone in 0.2% gold chloride for 5 min.
11. Fix in 5% sodium thiosulfate for 1 min.
12. Wash in dH₂O, dehydrate in alcohols then xylene and mount.