Myelin for Demyelination in brain tissue

1. Luxol fast blue for Myelin – using ammonia and alcohol as differentiators

Réactifs – source commerciale inconnue, sauf indication contraire :

• Mix and filter. Keeps indefinitely. Can be reused.

• Add this just before use – 5 to 10 drops of 10% acetic acid per 50 mL of stain.

Méthode:

1. De-wax sections to 95% alcohol.
2. Stain 3 – 4 hours in 0.1% Luxol fast blue at 60°C (waterbath/oven) or overnight at 37°C.
3. Remove excess stain with 95% alcohol.
4. Differentiate in 1% ammonia (with care); 5 – 10 seconds (until no more blue leaves section).
5. Bien laver à l'eau du robinet.
6. Stain in Cresyl fast violet at 60°C; 10 minutes.
7. Laver à l'eau.
8. Differentiate Nissl in 95% alcohol.
9. Laver à l'eau.
10. Déshydrater, clarifier et monter.

Résultats:

Myelin – blue

Nissl – purple

Nuclei – violet

2. Luxol fast blue for Myelin – using lithium carbonate and alcohol as differentiators

Réactifs – source commerciale inconnue, sauf indication contraire :

Méthode:

1. De-wax sections to 95% alcohol.
2. 1% Luxol fast blue at 60°C (waterbath/oven) for 6 hours, or overnight.
3. Rincer à l'alcool 70%.
4. Rincer à l'eau.
5. Differentiate in 0.05% Lithium carbonate for 15 minutes, until myelin is sharply differentiated macroscopically.
6. Rincer à l'eau.
7. Continue differentiation by rinsing in 70% alcohol.
8. Wash thoroughly in water.
9. 1% Cresyl violet (use pre-warmed) at 60°C for 5 minutes.
10. Differentiate in 95% alcohol until stain stops running.
11. Déshydrater, clarifier et monter.

Résultats:

Myelin and Erythrocytes – deep blue/purple

Nuclei and Nissl substance – purple/blue

3. Luxol fast blue – modified Klüver’s for Myelin

Réactifs – source commerciale inconnue, sauf indication contraire :

• Add solutions in the order above, mix with a magnetic stirrer for 1 hour.
• Filter solution using filter paper, store in closed container, stable for 1 year.

• Add solutions in the order above, mix with a magnetic stirrer.
• Filter solution using filter paper, store in closed container, stable for 1 year.

• Cover tightly with aluminium foil and stir with a magnetic stirrer for 1 hour.

Méthode:

1. De-wax sections to 95% ethanol:

• 4 minutes in xylene
• 2 minutes in xylene
• 1 minute in xylene
• 1 minute in 100% ethanol
• 1 minute in 100 % ethanol
• 1 minute in 95% ethanol

2. Submerge sections in Luxol fast blue solution using a sealable container. To protect against possible evaporation, seal the container with parafilm. Incubate at 60°C in a water bath overnight.
3. Rinse in 95% ethanol.
4. Rincer à l'eau distillée.
5. Differentiate slides in lithium carbonate solution for 10 seconds. Agitate constantly.
6. Continue differentiation in 2 changes of 70% ethanol for 10 seconds each. Agitate constantly.
7. Laver à l'eau distillée.
8. If required repeat steps 5-7 until there is a sharp contrast between the blue of the white matter and the colorless grey matter. As a rule of thumb repeat differentiation one time.
9. Rinse slides in 70% ethanol.
10. Incubate slides for 2 minutes in 0.3% Eosin.
11. Wash in distilled water thoroughly.
12. Incubate sections in 0.25% Cresyl Violet solution for 30 seconds.
13. Rinse slides in distilled water.
14. Dehydrate slides in 95% Ethanol for 10 seconds then 100% Ethanol for 10 seconds followed by 1 minute in clean xylene.
15. Coverslip slides.

Résultats:

Myelinated fibers – Blue

Nerve cells – Purple

Clause de non-responsabilité:

Ces méthodes sont fournies à titre indicatif uniquement. Les laboratoires qui souhaitent mettre en œuvre ces méthodes doivent procéder à une validation interne avant utilisation. Le RCPAQAP ne fait aucune déclaration ni ne donne aucune garantie quant à l'exactitude ou aux performances de ces méthodes.