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
| Reagent | Time | Technical Notes |
| 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.
|
| 95% alcohol | 1 change | - Rinse to remove excess stain
|
| Distilled water | 1 change | |
| *Lithium carbonate solution | 10 seconds | - Used for differentiation.
- Be careful not to over-differentiate.
|
*70% alcohol
| 2 changes | - Rinse to further differentiate and remove the 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 water | 3 changes |
|
| 70% alcohol | Differentiate until the background is colorless
| - Check microscopically for the endpoint.
|
Post-staining procedure: The tissue section should be dehydrated through graded alcohols, beginning with 95%. They were followed by 2 changes of xylene and then a coverslip.
Expected Results:
- Nissl substance - Dark blue to purple
- Nuclei - Dark blue to purple
- Myelin - Blue
