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
• Types of LFB
• Important considerations regarding LFB staining
• Example of degrees of differentiation

Staining procedure

1	Deparaffinization	Xylene	3 changes, 10 minuntes each
2	Removal of xylene	100% ethanol	3 changes, 5 minutes each
3	Hydration	95% ethanol	5 minutes
4	Staining ＊1	0.1% luxol fast blue solution	Overnight, 50℃
5	Rinsing	95% ethanol	1 minute
6	Washing ＊2	Running tap water
7	Differentiation ＊3	0.05% lithium carbonate	5 seconds to 20 seconds
8	Differentiation	70% ethanol	3 changes. 1 minute each in the first and second dish. Leave sections in the third dish for approximately 1 to 10 minutes.
9	Microscopic check		Repeat step ⑦ & ⑧, until gray matter become white.
10	Washing	Running tap water
11	Dehydration	70%, 95% ethanol	5 minutes each
12	Dehydration	100% ehtanol	3 changes, 5 minutes each
13	Clearing	Xylene	3 changes, 10 minuntes each
14	Coverslipping

0.1% Luxol fast blue solution (Filter before use)

Luxol fast blue	1g
95% ethanol	1000ml
10% acetic acid solution	5ml

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 name	Manufacturer	Availability
Luxol fast blue MBSN （Solvent blue 38）	EM SCIENCE	Out of stock
Solvvent blue 38 （Luxol fast blue MBSN）	SIGMA	Available commercially

Important considerations regarding LFB staining

＊1 Staining

• After LFB staining, return the specimen to room temperature and progress to the next procedure.

＊2 Washing

• Differentiate one section at a time in a dish of the differentiating solution.
• Stir the section in the solution with a dissecting needle.

＊3 Differentiation

• Differentiate one section at a time in a dish of the differentiating solution.
• Stir the section in the solution with a dissecting needle.
• The first dish quickly becomes dirty. Therefore, replace it with a new solution when its color darkens.
• Leave the section in the third dish until gray matter become colorless.
• Shake the third dish during the process.
• Step ⑦ & ⑧ may be repeated if more differentiaion is needed.
• Too much or too little differentiation produces false positives and false negatives, respectively. Care should be taken during the process.

⑥ 〜 ⑨ Experiment video

⑥ 〜 ⑨ Commentary illustrations

Important considerations regarding differentiation

• If too much color is lost in step ⑥, then this is irreparable and differentiation must be carefully conducted. Slowly and thoroughly remove the color in step ⑦.
• Repeat steps ⑥–⑧ until the cortex turns white. During this process, take care to avoid removing more color than necessary, which would end up removing the main pathological structures.

Examples of degrees of differentiation

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

Overdifferentiation

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.

Underdifferentiation

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