Rabbit anti p2x7

Total protein was extracted for western blotting analysis. The DRG tissues were homogenized in lysis buffer by mechanical disruption and incubated on ice for 50 min. After that, the lysates were centrifuged at 12,000 ×g and 4°C for 10 min. The supernatants were harvested to measure the protein concentrations using a bicinchoninic acid assay reagent kit and then preserved at −20°C for later use. After being diluted with the same amount of sample buffer (250 mmol/L Tris-Cl, 200 mmol/L dithiothreitol, 10% SDS, 0.5% bromophenol blue, and 50% glycerol), the supernatants were heated to 95°C for 10 min for denaturation. Supernatant samples containing 20 μg proteins were separated by 10% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked in 5% skim milk dissolved in buffer [10 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, and 0.05% Tween-20] for 1 h at room temperature and then incubated with the primary antibodies (rabbit anti-P2X₇, 1 : 1000, Abcam; mouse anti-β-actin, Sigma-Aldrich) overnight at 4°C, followed by incubation with an HRP-conjugated secondary antibody (1 : 5000, Beijing Zhongshan Biotech Co.) at room temperature. The band densities were determined using Image J Software and normalized to each β-actin internal control.

Immunohistochemistry was performed as follows. The rats’ DRG were removed and placed in 4 % paraformaldehyde for 2 h at room temperature. The DRG were washed in 0.1 M PBS prior to incubation in 20 % sucrose in PBS overnight. Then, 12-μm-thick sections were cut using a cryostat (Leica) and mounted onto the slides. The sections were washed in PBS and incubated in blocking solution containing 3 % bovine serum albumin (BSA) in PBS with 0.3 % Triton X-100 for 30 min at room temperature. Primary antibodies against glial fibrillary acidic protein (GFAP) (chicken anti-GFAP, Abcam) and P2X₇ (rabbit anti-P2X₇, Abcam) were diluted 1:100 in PBS containing 1 % BSA and incubated overnight at 4 °C. The sections were washed in PBS and incubated with the secondary antibody [goat anti-rabbit TRITC (tetraethyl rhodamine isothiocyanate) (Jackson ImmunoResearch Inc., West Grove, PA, USA) and goat anti-chicken FITC (Beijing Zhongshan Biotech CO.)] diluted 1:200 in PBS for 45 min at 37 °C. Finally, the sections were washed in PBS. Controls omitted the primary antibody. The sections were imaged using a fluorescence microscope (Olympus, Tokyo, Japan). Data were collected from six animals in each group. Five fields randomly selected that contained approximately 50 neurons each were analyzed from each animal and averaged.

Immunofluorescence was performed to measure the coexpression quantities of P2X₇ and glial fibrillary acidic protein (GFAP) in the DRG of DNP rats. The DRG of the rats were removed and fixed in 4% PFA for 2 h at room temperature. The DRG or sections were washed three times with 0.1 M PBS before the implementation of the following steps. DRG were incubated in 20% sucrose in PBS overnight, and after that, they were cut into 12 μm thick sections using a cryostat and mounted onto the slides. Then the DRG were blocked and permeabilized in PBS containing 3% BSA and 0.3% Triton X-100 for 30 min at room temperature. The sections were incubated with primary antibodies against GFAP (chicken anti-GFAP, Abcam) and P2X₇ (rabbit anti-P2X₇, Abcam) 1 : 100 diluted in PBS containing 1% BSA overnight at 4°C, after which they were incubated with the secondary antibodies [goat anti-rabbit TRITC (Jackson ImmunoResearch Inc., West Grove, PA, USA) and goat anti-chicken FITC (Beijing Zhongshan Biotech Co.)] 1 : 200 diluted in PBS for 45 min at 37°C. Finally, coverslips were mounted on slides and images were taken with a fluorescence microscope (Olympus, Tokyo, Japan).