Immunoblot polyvinylidene difluoride membranes

After the behavioral test, the SNs of the rats were rapidly dissected out and homogenized in lysis buffer (Beyotime Inst. Biotech, Beijing, China). The microglial cells were collected and lysed with a lysis buffer. Supernatants were collected and the protein concentrations were measured using a bicinchoninic acid protein assay kit (Beyotime Inst. Biotech). A total of 30 µg of protein was subjected to 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto immunoblot polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After blocking with 5% nonfat milk, the blots were incubated overnight at 4 °C with primary antibodies against iNOS (1:2000), COX-2 (1:1000), OX-42 (1:1000), TH (1:1000) (Abcam), phosphor-ERK1/2 (1:2000), ERK1/2 (1:2000), phosphor-p38 (1:2000), p38 (1:1000), phosphor-JNK (1:1000), JNK (1:2000), phosphor-NF-κB p65 (1:1000), NF-κB p65 (1:1000) (Cell Signaling Technology, Danvers, MA, USA), and β-actin (1:2000) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). After this, the blots were incubated with a horseradish peroxidase-labeled secondary goat anti-rabbit (1:2000; Santa Cruz, CA, USA) or rabbit anti-goat antibody (1:2000; Santa Cruz, CA, USA) for 1 h at room temperature.