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
Show moreDeparaffinize and hydrate sections to dH2O. Wash 3X for 3 min. in dH2O.
Place sections in 50 ml 10% silver nitrate in dark at 37ºC for 30 min.
Wash 3 times for 3 min in dH2O.
Add concentrated ammonium hydroxide dropwise with stirring to the silver nitrate solution reserved from step 2.
Incubate sections in this solution for 15 min at 37ºC.
Wash sections in 0.1% ammonium hydroxide 3 times for 2 min at room temperature.
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.
Stain sections in this solution for 10 min until they turn black.
Wash in 0.1% ammonium hydroxide 3 times for 2 min and dH2O 3 times for 2 min.
Tone in 0.2% gold chloride for 5 min.
Fix in 5% sodium thiosulfate for 1 min.
Wash in dH2O, dehydrate in alcohols then xylene and mount.
Fig 1. Example of axonal silver staining Showing axons stained using Bielshchowsky's silver stain. 8 µm thick paraformaldehyde paraffin embedded of mouse cerebellum sections.