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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
- 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.
- 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.
- 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.
- Examine under a light microscope. A 100X oil immersion lens is recommended for detailed images used to calculate g-ratios.