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Luxol Fast Blue (LFB) - Cresyl Violet Staining - Staining Protocol

Sample type required: Deparaffinized and rehydrated tissue section (10-15 μ) on positively (+) charged slides
Preferred fixative: 10% neutral buffered formalin (NBF)
Control: Spinal cord or medulla

ReagentTimeTechnical Notes
Luxol fast blue (LFB) solutionOvernight in a 58° C oven
  • Be sure to cap/seal the coplin jar to prevent evaporation of the solution.
95% alcohol1 change
  • Rinse to remove excess stain
Distilled water1 change
  • Rinse
*Lithium carbonate solution10 seconds
  • Used for differentiation.
  • Be careful not to over-differentiate.
*70% alcohol
2 changes
  • Rinse to further differentiate and remove lithium carbonate solution.
Repeat the previous 2 steps denoted by the (*)Until there is a sharp contrast between the blue of the white-matter and the colorless gray-matter.
  • Check microscopically for differentiation.
  • Be careful not to over-differentiate.
    0.5% cresyl violet
    5 minutes
      Distilled water3 changes
        70% alcoholDifferentiate until background is colorless
        • Check microscopically for end point.
        Post staining procedure: Tissue section should be dehydrated with 95% and absolute alcohols. Follow with 2 changes of xylene and then coverslip.
        Expected Results:
        • Nissl substance - Dark blue to purple
        • Nuclei - Dark blue to purple
        • Myelin - Blue