Bielschowsky Silver Staining - Staining Protocol

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Bielschowsky Silver Staining - Staining Protocol

Sample type required: Deparaffinized and rehydrated tissue section (6-8 μ) on positively (+) charged slides
Preferred fixative: 10% neutral buffered formalin (NBF)
Control: Cerebral cortex of a known case of Alzheimer's disease
ReagentTimeTechnical Notes
20% silver nitrate solution15 minutes in 30° C oven
  • Preheat the solution to 60° C in an oven or waterbath.
  • Add slides to the warm solution for 15 minutes in 37° C oven.
Distilled water3 changesRinse one slide at a time and place in a clean, dry staining jar or rack.
Working ammoniacal silver5 to 30 minutes in 37°C oven
  • Make fresh right before use.
Working Formalin-based developer solution1 - 5 minutes
  • Check silver development on nerve fibers under the microscope.
  • Background should be golden brown.
  • Nerve fibers should be turning black.
Fresh ammonia water wash
Distilled water wash 3 changes
5% sodium thiosulfate2 minutes
Fresh tap waterWash well
Post staining procedure: Tissue section should be dehydrated with graded alcohols beginning with 95%. Follow with 2 changes of xylene and then coverslip.
Expected results:
  • Axons and neurofibrillary tangles - Black
  • Plaque and amyloid - Brown to dark brown
  • Background - Golden brown
  • Slides should not be handled with metal forceps or any metallic substance as a black precipitant will form from exposure to silver solution.

Bielschowsky stain of brain section showing neurofibrils