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Bielschowsky's silver stain

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

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Last edited Mon 29 Aug 2022

Stage 1 - Bielschowsky’s silver stain

Steps

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 3 times 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 3 times 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 3 times for 2 min and dH2O 3 times 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.

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.