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

Abstract

Source: Ghnenis, A. B. et al., Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology. J. Vis. Exp. (2018)

This video demonstrates the staining technique of resin-embedded, osmium tetroxide-treated rat peripheral nerve sections using toluidine blue. The stained nerve sections help visualize the fine structures of peripheral nerves.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Toluidine Blue Staining

1. Prepare 1% solution of toluidine blue by dissolving 2 g of sodium borate in 100 mL of deionized water, then add 1 g of toluidine blue and stir until dissolved. Filter the solution using filter paper (pore size: 11 µm) and keep the solution in an opaque bottle at room temperature for up to two weeks.
2. Using a plastic pipette or micro pipettor, add a drop of toluidine blue solution on the top of the nerve sections and leave for 20-30 s.
3. Rinse off all toluidine blue solution excess by gently dipping the slides into deionized water jar and repeat 3-4 times until sections are clear. Dry the slides at least 15 min at 60 °C or overnight at room temperature and then cover the sections with a coverslip using regular mounting medium. Examine the mounted slides immediately or store at room temperature.
4. Examine under a light microscope. A 100X oil immersion lens is recommended for detailed images used to calculate g-ratios.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Toluidine Blue O	Sigma-Aldrich	T3260
Sodium Tetraborate Decahydrate	Acros Organics	205950010
Microscope Cover Glass	Fisher Scientific	12545102
VWR Micro Slides, Superfrost Plus	VWR	48311-703
100 Watt Oven	Millipore	6350115
Whatman Filter Paper	Sigma-Aldrich	WHA10010155
Olympus fluorescence microscope			Dual CCD Color and Monochrome Camera, DP80
3 mL plastic pipette	Sigma-Aldrich	Z331740