Bielschowsky’s Silver Stain for Axons

By Paul Polak and Douglas Feinstein *

  1. Deparaffinize and hydrate sections to dH20.  Wash 3x 3 min. in dH20.
  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 3 min. in dH20.
  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 6.
  6. Wash sections in 0.1% ammonium hydroxide 3x 2 min. at room temperature.
  7. Add 350 μl developer solution (0.2 ml 37% formaldehyde, 12 ml dH20, 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 2 min. and dH20 3x 2 min.
  10. Tone in 0.2% gold chloride for 5 min.
  11. Fix in 5% sodium thiosulfate for 1 min.
  12. Wash in dH20, dehydrate in alcohols then xylene and mount.    

Example of axonal silver staining: 8 μm thick paraffin embedded paraformaldehyde fixed sections of EAE mouse brains

silver stain axon copy II

University of Illinois at Chicago