⑫ Staining

Luxol fast blue (LFB) staining

Myelin sheaths are stained blue with Luxol fast blue.

Luxol fast blue is used for Klüver-Barrera (KB) staining

Staining procedure

1DeparaffinizationXylene3 changes, 10 minuntes each
2Removal of xylene100% ethanol3 changes, 5 minutes each
3Hydration95% ethanol5 minutes
4Staining *10.1% luxol fast blue solutionOvernight, 50℃
5Rinsing95% ethanol1 minute
6Washing *2Running tap water 
7Differentiation *30.05% lithium carbonate5 seconds to 20 seconds
8Differentiation70% ethanol3 changes. 1 minute each in the first and second dish. Leave sections in the third dish for approximately 1 to 10 minutes.
9Microscopic check Repeat step ⑦ & ⑧, until gray matter become white.
10WashingRunning tap water 
11Dehydration70%, 95% ethanol5 minutes each
12Dehydration100% ehtanol3 changes, 5 minutes each
13ClearingXylene3 changes, 10 minuntes each

0.1% Luxol fast blue solution (Filter before use)

Luxol fast blue1g
95% ethanol1000ml
10% acetic acid solution5ml

Types of LFB

Two types of LFB are currently used in the Laboratory of Neuropathology. Whichever product is used, the same stained chromatic image can ultimately be obtained through differentiation and observation of the specimen under a microscope.

Product nameManufacturerAvailability
Luxol fast blue MBSN
(Solvent blue 38)
EM SCIENCEOut of stock
Solvvent blue 38
(Luxol fast blue MBSN)
SIGMAAvailable commercially

Important considerations regarding LFB staining

*1 Staining

*2 Washing

Differentiate one section at a time in a dish of the differentiating solution.

*3 Differentiation

⑥ 〜 ⑨ Experiment video

⑥ 〜 ⑨ Commentary illustrations

Important considerations regarding differentiation

Examples of degrees of differentiation

Amyotrophic lateral sclerosis of the lateral funiculus of the spinal cord: The breakdown of the myelin sheath.


The background is completely colorless, and it is difficult to find phagocytosis by macrophages.

Moderate differentiation

We can see the breakdown products of myelin sheathes engulfed by macrophages.


The entire specimen is blue. It is impossible to distinguish the breakdown products of the myelyn sheath.