Sample type required: Deparaffinized and rehydrated tissue section (10-15 μm) on positively (+) charged slides
Preferred fixative: 10% neutral buffered formalin (NBF)
Control: Spinal cord or medulla
Luxol fast blue (LFB) solution | Overnight in a 58° C oven | - Be sure to cap/seal the coplin jar to prevent evaporation of the solution.
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95% alcohol | 1 change | - Rinse to remove excess stain
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Distilled water | 1 change | |
*Lithium carbonate solution | 10 seconds | - Used for differentiation.
- Be careful not to over-differentiate.
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*70% alcohol
| 2 changes | - Rinse to further differentiate and remove lithium carbonate solution.
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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.
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0.5% cresyl violet
| 5 minutes |
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Distilled water | 3 changes |
|
70% alcohol | Differentiate until background is colorless
| - Check microscopically for end point.
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Post staining procedure: Tissue section should be dehydrated through graded alcohols beginning with 95%. Follow with 2 changes of xylene and then coverslip.
Expected Results:
- Nissl substance - Dark blue to purple
- Nuclei - Dark blue to purple
- Myelin - Blue