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Luxol Fast Blue (LFB) Staining - Staining Protocol

Sample type required: Deparaffinized and rehydrated tissue section (10 μ) on positively (+) charged slides
Preferred fixative: 10% neutral buffered formalin (NBF)
Control: Normal cerebral cortex, spinal cord, or medulla
Reagent TimeTechnical Notes
Luxol fast blue (LFB) solutionOvernight in 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
  • Use for differentiation.
  • Be careful not to over-differentiate.
*70% alcohol2 changes
  • Rinse to further differentiate and remove lithium carbonate solution.
*Distilled water3 changes
  • Wash to remove alcohol.
Repeat the previous 3 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.

Post staining procedure: Tissue section should be dehydrated with 95% and absolute alcohols. Follow with 2 changes of xylene and then coverslip.
Expected results:
  • Myelin - Blue
  • Background - Colorless

Image courtesy of Wikimedia Commons.