Myelin is a membrane that wraps around the axon of a nerve cell, i.e., myelin consists of the cell membrane of myelinating cells; current research pays attention to the antigenicity of myelin components, such as: myelin basic protein (MBP), myelin-associated glycoproteins (MAG), myelin oligodendrocyte glycoproteins (MOG), and so on.

Operation method

LFB myelin staining method

Principle

LuxolFastBlue (LFB) belongs to copper-phthalocyanine dyes, and has the staining property of binding to myelin phospholipids in alcoholic solution. The application of LFB myelin staining can show the myelin structure of nerve tissue very well.

Materials and Instruments

Myelin sheath
LFB solution 0.05% lithium carbonate solution 0.25% tar violet solution
Filter

Move

1、After the slices were cleaned by distilled water, they were put into 0.1% LFB solution and sealed and dipped at 60℃ for 8-16h.

2, after cleaning by distilled water, into 95% alcohol.

3, to 0.05% lithium carbonate aqueous solution color separation for more than 10s.

4, by 70% alcohol to continue color separation, until the microscope to observe the gray, white matter distinction is clear.

5、 After washing the film with distilled water, the film was re-stained with 0.25% tar violet solution plus a few drops of glacial acetic acid dye solution for 10min.

6, 70% alcohol will be re-staining color until the cell nucleus and Nippon body is red.

(7) Filter paper was stained with excess liquid, and rinsed and dehydrated into n-butanol for 2 times, with each dehydration time of 3-5min.

(8) Xylene transparent, neutral gum sealing film. The myelin sheath is bright blue, and the nucleus is dark blue.

Caveat

1、Sections, if paraffin sections, should be deparaffinized in xylene first, then to anhydrous ethanol.

2， 0.05% lithium carbonate aqueous solution color separation should be carried out under the microscope, until the background is gray. 70% alcohol again color separation, until the background is colorless.

Common Problems

1、After LFB staining, the cells can be restained with tar violet solution or hematoxylin-eosin, which can better show the morphology of the cells.

2、After re-staining, it should be rinsed and dehydrated in n-butanol, so that the staining effect is better.

3, solution preparation: Rauch Firm Blue 1g, 95% alcohol 100ml, 10% glacial acetic acid 0.5ml. mixing and filtration, room temperature storage.

Source: Practical Experimental Techniques in Neurobiology

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