Overview
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
- 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.
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Materials
| 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 |
