Rabbit anti trpv1

SD rats were divided into the control and CFA groups, plantar injection three days later, and fresh brain tissues containing the CSF-contacting nucleus were collected. Ten times the tissue weight of RIPA lysis buffer (Beyotime, China) and 1/100 of the lysate volume of PMSF (Beyotime, China) was added. After the tissues were homogenized and centrifuged at 4°C for 15 min at 12,000 rpm, the supernatant was collected, and the total protein concentration of each sample was determined by a BCA protein assay kit (Beyotime, China). Then, the proteins were separated by 8% SDS-PAGE. After electrophoresis, the proteins were transferred to PVDF membranes, which were blocked with 5% skim milk powder for 2 h and incubated with rabbit anti-TRPV1 (1:2000, Novus Biologicals), mouse anti-GABA_B1 (1:500, Abcam) and mouse anti-GAPDH (1:2000, Proteintech) antibodies at 4°C overnight. After half an hour at room temperature, the PVDF membranes were rinsed three times with TBST (TBS containing 1% Tween-20) and incubated with corresponding HRP-conjugated secondary antibodies (1:2000, Beyotime, China) for 2 h. Then, the membranes were rinsed six times with TBST. The protein bands were detected by a chemiluminescent reagent (Beyotime, China), and the relative grayscale values were analyzed using ImageJ software.