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Luxol Fast Blue Staining Protocol for Myelin
Description: This is for staining of myelin/myelinated axons on formalin-fixed, paraffin-embedded brain and spinal cord tissue sections, as well as frozen sections. The myelin, including phospholipids, will be stained blue to green, and the neurons will be stained violet. This stain is commonly used for identifying the basic neuronal structure in brain or spinal cord tissue.
Fixation:
10% formalin.
Section:
paraffin sections at 5-10 um. Frozen sections at 20-30um.
Solutions and Reagents:
0.1% Luxol fast blue
solution:
Luxol fast blue, MBS
-------------------------- 0.1 gm
Ethyl Alcohol, 95%
---------------------------- 100 ml
Glacial acetic acid
---------------------------- 0.5 ml
0.1% Cresyl echt violet solution:
Cresyl echt violet (cresyl fast
violet) ------- 0.1 gm
Distilled water
-------------------------------- 100 ml
Add 10 drops of glacial acetic
acid just before use and filter.
0.05% Lithium carbonate solution:
Lithium carbonate
------------------------------- 0.05 gm
Distilled water
----------------------------------- 100 ml
Procedure:
1. Deparaffinize and hydrate to 95% ethyl alcohol (Frozen/vibratome
sections may need to have de-fat step: place sections directly into
1:1 alcohol/chloroform for a few hours/overnight and then hydrate
back 95% ethyl alcohol).
2. Leave in luxol fast blue solution in 56 C oven overnight (for
frozen sections, not longer than 16 hours).
3. Rinse off excess
stain with 95% ethyl alcohol.
4. Rinse in distilled
water.
5. Differentiate the
slides in the lithium carbonate solution for 30 seconds.
6. Continue
differentiation in the 70% ethyl alcohol for 30 seconds.
7. Rinse in distilled water.
8. Check microscopically to see if gray matter is clear and
white matter sharply defined.
9. Repeat the
differentiation steps (step 5-7) if necessary.
10.
When
differentiation is complete, place in distilled water.
11.
Counterstain in the cresyl violet solution for 30-40 seconds.
12.
Rinse in distilled
water (don’t rinse in 70% alcohol which will clear off fast blue
staining).
13.
Differentiate the
slides in 95% ethyl alcohol for 5 minutes (check microscopically).
14.
100% alcohol 2x5
min.
15.
Xylene 2x5 min.
16.
Mount with resinous medium.
Results:
Myelin,
including phospholipids ------------------ blue to green
Neuron
---------------------------------------------- pink to violet
Positive Controls:
Brain, spinal cord.