Anatomical Pathology Special Stains and Immunohistochemistry
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Home > Myelin for Demyelination in brain tissue

Myelin for Demyelination in brain tissue

Aim of special stain:
Demonstrating the myelin sheath, exhibiting optimal contrast between grey and white matter
Diagnostic applications:
Diagnosis of demyelination in brain tissue such as MS (multiple sclerosis)
Material supplied for surveys:
Brain tissue from a known case of MS, cut at 3-7µm (some laboratories may prefer thicker sections)
Recommended control:
Brain tissue with demyelination, as well as areas of normal brain tissue to act as an internal control

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

Reagents – commercial source unknown, unless specified:

1. 10% Acetic Acid
a) Glacial acetic acid 10 mL
b) Distilled water 90 mL
2. 0.1% Luxol fast blue
a) Luxol fast blue, MBS (Dye) 0.1 g
b) 95% Ethyl alcohol 100 mL
c) 10% Acetic acid 0.5 mL
  • Mix and filter. Keeps indefinitely. Can be reused.
3. 0.1% Cresyl fast violet – prepare fresh
a) Cresyl fast violet (Dye) 0.1 g – Cresyl violet acetate
b) Distilled water 100 mL
  • Add this just before use – 5 to 10 drops of 10% acetic acid per 50 mL of stain.
4. 1% Ammonia
a) Ammonia 1.0 g
b) Distilled water 100 mL
5. 95% Alcohol
a) 100% Alcohol 950 mL
b) Distilled water 50 mL

Method:

  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. Wash well in tap water.
  6. Stain in Cresyl fast violet at 60°C; 10 minutes.
  7. Wash in water.
  8. Differentiate Nissl in 95% alcohol.
  9. Wash in water.
  10. Dehydrate, clear and mount.

Results:

Myelin – blue

Nissl – purple

Nuclei – violet

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

Reagents – commercial source unknown, unless specified:

1. 10% Acetic Acid
a) Glacial acetic acid 10 mL
b) Distilled water 90 mL
2. 0.1% Luxol fast blue (stable for 18 months)
a) Luxol fast blue 0.1 g
b) 95% Alcohol 100 mL
c) 10% Acetic acid 0.1 mL
3. 0.1% Cresyl violet – (stable for 2 years)
a) Cresyl violet 0.1 g
b) Distilled water 100 mL
c) 10% Acetic acid 1 mL
4. 0.05% Lithium carbonate (stable for 2 years)
a) Lithium carbonate 0.1 g
b) Distilled water 200 mL
5. 95% Alcohol
a) 100% Alcohol 950 mL
b) Distilled water 50 mL

Method:

  1. De-wax sections to 95% alcohol.
  2. 1% Luxol fast blue at 60°C (waterbath/oven) for 6 hours, or overnight.
  3. Rinse in 70% alcohol.
  4. Rinse in water.
  5. Differentiate in 0.05% Lithium carbonate for 15 minutes, until myelin is sharply differentiated macroscopically.
  6. Rinse in water.
  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. Dehydrate, clear and mount.

Results:

Myelin and Erythrocytes – deep blue/purple

 

Nuclei and Nissl substance – purple/blue

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

Reagents – commercial source unknown, unless specified:

1. 10% Acetic Acid
a) Glacial acetic acid 10 mL
b) Distilled water 90 mL
2. 0.1% Luxol fast blue – Du Pont
a) Luxol fast blue powder 0.1 g
b) 95% Ethanol 100 mL
c) 10% Acetic acid 0.5 mL
  • 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.
3. 0.25% Cresyl Echt violet – Pharmaceutical Laboratories National Aniline Division
a) Cresyl Echt violet 0.25 g
b) Distilled water 100 mL
c) 10% Acetic acid 1 mL
  • Add solutions in the order above, mix with a magnetic stirrer.
  • Filter solution using filter paper, store in closed container, stable for 1 year.
4. 0.05% Lithium carbonate – Merck Pty Ltd – store in room temperature
a) Lithium carbonate 0.5 g
b) Distilled water 1 L
5. 0.3% Eosin in 70% Ethanol – store in room temperature
a) Eosin powder 6 g
b) 70% Ethanol 2 L
  • Cover tightly with aluminium foil and stir with a magnetic stirrer for 1 hour.
6. 95% Ethanol
a) 100% Ethanol 950 mL
b) Distilled water 50 mL
7. 70% Ethanol
a) 100% Ethanol 700 mL
b) Distilled water 300 mL

Method:

  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
  1. 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.
  2. Rinse in 95% ethanol.
  3. Rinse in distilled water.
  4. Differentiate slides in lithium carbonate solution for 10 seconds. Agitate constantly.
  5. Continue differentiation in 2 changes of 70% ethanol for 10 seconds each. Agitate constantly.
  6. Wash in distilled water.
  7. 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.
  8. Rinse slides in 70% ethanol.
  9. Incubate slides for 2 minutes in 0.3% Eosin.
  10. Wash in distilled water thoroughly.
  11. Incubate sections in 0.25% Cresyl Violet solution for 30 seconds.
  12. Rinse slides in distilled water.
  13. Dehydrate slides in 95% Ethanol for 10 seconds then 100% Ethanol for 10 seconds followed by 1 minute in clean xylene.
  14. Coverslip slides.

Results:

Myelinated fibers – Blue

Nerve cells – Purple

Disclaimer:

These methods are intended as a guide only. Laboratories that wish to implement these methods should perform internal validation prior to use. The RCPAQAP does not make any claim or warranty for the accuracy or performance of these methods.

References

  1. 2021 RCPAQAP Neuropathology Technical Survey 1.

  2. 2025 RCPAQAP Neuropathology Technical Survey 1.

  3. Nestor. Scott L, Techniques in Neuropathology in Bancroft J, Gamble M. Theory and Practice of Histology Techniques.  6th Edition.2008, p376-378. Churchill Livingstone.

  4. Ralis HM, Beesley RA, Ralis ZA. Techniques in Neurohistology. London: Butterworth and Co, 1973: 110-2.

  5. Klűver H, Barrera BS. A method for the combined staining of cells and fibres in the nervous system. Journal of Neuropathology and Experimental Neurology, 1953; X11: 400-4.

  6. Bancroft JD, Stevens A. Theory and Practice of Histological techniques, 4th ed. 1996.

  7. Sheehan D, Hrapchak B, Theory and practice of Histotechnology, 2nd ed, 1980, pp262-264, Battelle Press, Ohio.

  8. Crookham,J, Dapson,R, Hazardous Chemicals in the Histopathology Laboratory, 2nd ed, 1991, Anatech.

  9. Bancroft J, Gamble M. Theory and Practice of Histology Techniques. 6th Edition. 2008, p137-138. Churchill Livingstone.

Last updated on January 30, 2026

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