Rabbit anti gap 43

Sciatic nerves and left L₃-L₅ DRGs were dissected and homogenized rapidly on ice at the time point of one week and two weeks after sciatic injury. The protein concentration of each sample was determined by Bradford assay (Sangon Biotech, China). Twenty micrograms homogenate of DRGs and 30 μg homogenate of sciatic nerves were uploaded onto 10% acrylamide gel for sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein was then transferred onto polyvinylidene fluoride membranes. Afterward, the membranes were blocked with 5% no-fat milk solution and then incubated with mouse anti-TRPV1 (1:4000, Biosensis, USA), mouse anti-GFAP (1:2500, Novus Biologicals, USA), or rabbit anti-GAP-43 (1:5000, Bioworld, USA) overnight at 4°C. On the second day, the membranes were washed with 0.1% Tris-buffered saline and Tween 20, 3× 5 min and then incubated for 1 h in horseradish peroxidase (HRP)-conjugated goat antimouse IgG (1:5000, Sangon Biotech, China) or HRP-conjugated goat antirabbit IgG (1:5000, Sangon Biotech, China). Finally, the protein bands were detected after incubated in Easy See Western Blot ECL reagent (Sangon Biotech, China) and exposed onto X-ray film in dark room. Meanwhile, rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:5000, Bioworld, USA) was used as a loading control.

Sciatic nerves were dissected rapidly at the time point of one week and two weeks after sciatic nerve transection and then were freshly embedded with optimal cutting temperature compound and fixed in the liquid nitrogen. Samples were stored at −80°C till use. Nerves were longitudinally cut into sections of 12 μm thickness. The slices were kept onto the glass slides, which were precoated with 0.1% polylysine. The sections were dried in the air completely and immersed into 4% paraformaldehyde for 12 h of fixation. Sections were first washed three times with 0.01 M phosphate-buffered solution (PBS) and incubated in 0.1% Triton for 10 min. After 1 h of incubation with 10% normal serum, two primary antibodies (mouse anti-GFAP (1:1000, Novus Biologicals, USA) and rabbit anti-GAP-43 (1:500, Bioworld, USA)) were applied onto slices, incubated overnight at 4°C. On the following day, the secondary antibodies of Alexa Fluor® conjugated with 488 or 594 (1:500, Life Technology, USA) were loaded onto slices after substantially washing with 0.01 M PBS. Finally, the sections were mounted with an antifade-mount medium (Life Technology). Immunofluorescence images of GFAP and GAP-43 were observed under upright immunofluorescence microscope (Olympus BX43, Japan).