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Holzer's Crystal Violet 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: Normal cerebral cortex
ReagentTimeTechnical Notes
Phospohmolybdic acid-alcohol3 minutes
  • Drain off excess fluid and place slides on a staining rack.
Absolute alcohol - chloroform mixture
  • Prepare fresh.
  • Cover each section with this mixture while still on the staining rack.
  • The tissue should become translucent.
Crystal violet stain30 seconds
  • Prepare fresh.
  • Add stain while the sections are still covered in absolute alcohol - chloroform.
10% potassium bromide1 minute
  • Drain slides of absolute alcohol-chloroform mixture and crystal violet stain.
  • Wash in potassium bromide.
  • Blot each section and allow them to thoroughly air dry.
Differentiating solution*30 seconds
  • Prepare fresh.
  • Differentiate each slide INDIVIDUALLY.
Xylene3 changes
  • Wash.
  • Check macroscopically to ensure that the background is pale or colorless.
  • Repeat the differentiating step(*) as needed to obtain this result.
    Post staining procedure: Tissue section should be coverlipped immediately.
    Expected results:
    Glial fibers - Purplish blue
    Background - Pale blue to colorless

    Glial cells stained with Holzer's crystal violet. Image courtesy of Wikimedia Commons.