Toluidine Blue Staining of Peripheral Nerve Sections: A Technique to Stain Myelinated Nerve Fibers For Peripheral Nerve Structure Visualization

JoVE Encyclopedia of Experiments

Biologische Technieken

0 weergaven • 2:50 min • July 8th, 2025

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Peripheral nerves consist of axons surrounded by Schwann cells that wrap around the axons with multiple layers of lipid-rich cell membranes, forming the myelin sheath.

To visualize peripheral nerves, begin with a glass slide carrying a fixed, resin-embedded, osmium tetroxide-treated transverse rat peripheral nerve section. Resin-embedded sections help maintain tissue morphology.

Osmium tetroxide treatment causes deposition of metallic osmium in the lipid layer of myelin sheath at the tissue site.

Pipette alkaline solution of toluidine blue over the tissue section. Dry the slide at an appropriate temperature. The alkaline conditions and heat facilitate toluidine blue penetration through the resin into the tissue section.

Toluidine blue, being a cationic dye, binds to the negatively-charged phosphate groups on DNA and also intercalates between the DNA base pairs in Schwann cell nuclei. In addition, the dye binds to RNA in the cytoplasm, outside the myelin sheath.

Add a mounting solution over the stained nerve section and cover with a coverslip to prepare the tissue for imaging. Under a light microscope, myelin sheath layers appear black with defined myelinated nerve fiber structures following osmium treatment, while the axons remain unstained. 

Schwann cell nuclei and cytoplasm appear different shades of blue due to metachromatic property of the dye, facilitating visualization of axon morphology and degree of myelination in peripheral nerves.

To stain the nerve sections, use a plastic pipette or micro pipettor to add a drop of toluidine blue solution on the top of the nerve sections. After 20 to 30 seconds, rinse off all toluidine blue solution excess, by gently dipping the slides into a jar of deionized water, repeating 3 to 4 times until sections are clear.

Dry the slides for at least 15 minutes at 60 degrees Celsius, or overnight at room temperature. After drying, add regular mounting medium, and cover the sections with a coverslip. Finally, examine the mounted sections under a light microscope. A 100X oil immersion lens is recommended for detailed images to calculate g-ratios.

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Laatst bijgewerkt: 22 augustus 2026