Anti il 1β 3a6

The hind paw tissues were collected from euthanized mice and shredded with surgical scissors in disposable culture dishes placed on dry ice. All tissue pieces (1 mm × 1 mm) were placed in 1.5 ml tube, added to1 ml RIPA lysis buffer (Santa Cruz) and then homogenized with a bead microtube homogenizer (Sigma-Aldrich) for 45 s at a speed of 5 m/s. Tubes were then kept on ice for 30 min and centrifuged at 12,000 g for 10 min at 4 °C. Afterwards, the supernatants were gathered in individual tubes and stored at − 80 °C. For Western blot detection, total paw-extracted proteins were quantified by a Pierce™ BCA Protein Assay Kit, and 80 μg of protein was loaded on a 5% concentrated gel and 10–12% segregated polyacrylamide gels. After 1 h of electrophoresis, the separated proteins were transferred to PVDF membranes (100 V, 60 min) and blocked with 5% skimmed milk for 1 h. Membranes were then incubated with anti-NLRP3 (D4D8T) (Cell Signaling Technology), anti-caspase-1 p10 (M-20) (Santa Cruz), anti-IL-1β (3A6) (Cell Signaling Technology) and anti-GAPDH antibodies (Abcam) at 4 °C overnight. The Goat anti-rabbit IgG H&L (HRP) was incubated with secondary antibodies at a concentration of 1:1000 (RT, 1 h) before reaction with Pierce™ ECL Plus Western Blotting Substrate. The greyscale values of the bands were quantified by Image-Pro Plus Version 7.0 software (Media Cybernetics).