Protocol for fixation, teasing and staining of sciatic nerves:

Fixation:

1. For adult sciatic nerves: Perform a transcardial perfusion with fresh cold 4% paraformaldehyde (PFA). Remove left and right sciatic nerves and place in cold 4% PFA at 4ºC for four hours.

2. For early postnatal nerves (P0-P10) sacrifice animal, remove sciatic nerves and place in fresh cold 4% PFA for 1.5 hours. (Note: May be necessary to adjust the duration of the fixation and/or concentration of the PFA for certain antigens.)

3. After fixation, remove PFA and immerse nerves in fresh cold PBS. Store sciatic nerves in PBS at 4ºC overnight and begin teasing the following day. Nerves may be stored at 4ºC in PBS for up to a few days before beginning to tease them.

Teasing:

4. Transfer fixed nerves to a 60mm diameter Petri dish less than half full with PBS.

5. Remove perineurial sheath by holding one end of the nerve segment with forceps and gently stripping away the sheath with a fine needle or a second set of forceps.

6. Subdivide the nerve segment into several fascicles with the fine needle. This requires lots of patience.

7. Using microdissection scissors, cut a small fascicle about 1 cm in length, containing approximately 50 nerve fibers, from the main nerve trunk.

8. Place a couple drops of water on a slide and transfer the small bundle of nerves to the slide.

9. Still holding one end of the nerve fibers with forceps, gently drag them across the slide. At the same time, use the long fine needle to ‘brush’ them out on the slide. Ideally, you should have as much separation between the individual nerves fibers as possible. Visualizing the individual nerves is best accomplished when there is space between each nerve. This requires time and patience.

10. Allow the slides to air dry overnight and then store slides at –80ºC or stain them immediately as follows.

Staining:

11. Permeabilize slides with fresh cold methanol for 15 minutes at -20ºC.

12. Wash with fresh 1X PBS 2 times, 5 minutes/wash, at RT.

13. Carefully dry slides and trace a small circle around the teased fibers with a PAP pen.

14. Block for 1 hour at RT. Blocking solution: PBS + 2.5% donkey serum, 2.5% BSA, 0.5% Triton X-100.

15. Dilute primary antibody in blocking solution prior to adding to slides. It typically requires only ~75uL of primary antibody to cover the TSN, providing a small enough circle has been made with the PAP pen.

16. Incubate in primary antibody overnight at 4ºC in a humidifying chamber.

17. Wash 3X with PBS: 10min, 10min, 5min.

18. Prepare secondary immediately before use. Incubate slides in secondary antibody for 1 hour at RT, in the dark. Note: may need to retrace circle around fibers with PAP pen before adding secondary.

19. Wash 4X with PBS, 5min each wash.

20. Wash 1X with dH20 for 5min. Dry slides thoroughly.

21. Mount with Citifluor (+Hoescht). Use ~30 uL of Citifluor for each slide, placing a drop directly on the teased fibers. Apply coverslip without creating any air bubbles, and seal the edges with clear nail polish.