Horseradish peroxidase hrp conjugated goat anti mouse igg

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.