Myelin for Demyelination in brain tissue

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

Reagen – sumber komersial tidak diketahui, melainkan dinyatakan:

• 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.

Kaedah:

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. Basuh dengan baik dalam air paip.
6. Stain in Cresyl fast violet at 60°C; 10 minutes.
7. Basuh dalam air.
8. Differentiate Nissl in 95% alcohol.
9. Basuh dalam air.
10. Dehidrasi, jelas dan lekapkan.

Keputusan:

Myelin – blue

Nissl – purple

Nuclei – violet

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

Reagen – sumber komersial tidak diketahui, melainkan dinyatakan:

Kaedah:

1. De-wax sections to 95% alcohol.
2. 1% Luxol fast blue at 60°C (waterbath/oven) for 6 hours, or overnight.
3. Bilas dalam alkohol 70%.
4. Bilas dalam air.
5. Differentiate in 0.05% Lithium carbonate for 15 minutes, until myelin is sharply differentiated macroscopically.
6. Bilas dalam air.
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. Dehidrasi, jelas dan lekapkan.

Keputusan:

Myelin and Erythrocytes – deep blue/purple

Nuclei and Nissl substance – purple/blue

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

Reagen – sumber komersial tidak diketahui, melainkan dinyatakan:

• 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.

Kaedah:

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. Bilas dalam air suling.
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. Basuh dalam air suling.
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.

Keputusan:

Myelinated fibers – Blue

Nerve cells – Purple

Penafian:

Kaedah ini bertujuan sebagai panduan sahaja. Makmal yang ingin melaksanakan kaedah ini harus melakukan pengesahan dalaman sebelum digunakan. RCPAQAP tidak membuat sebarang tuntutan atau jaminan untuk ketepatan atau prestasi kaedah ini.