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.