Anti p2x7r

Sciatic nerves were lysed in RIPA buffer (Solarbio) containing a protein phosphatase inhibitor cocktail. Proteins were separated by performing sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 10% resolving gels and then transferred onto polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk and then incubated with the following primary antibodies overnight at 4 °C: anti-P2X7R (1:1000, Abcam), anti-NLRP3 (1:500, Thermo Fisher), anti-IL-1β (1:1000, Abcam), and anti-Caspase-1 (1:1000, Abcam). Next, the membranes were washed and incubated with donkey anti-rabbit IgG or donkey anti-mouse IgG conjugated with HRP at a 1:5000 dilution (Thermo Fisher Scientific) for 1 h at room temperature. Finally, the immunoreactive protein bands were detected using a Gel Doc imaging system (Bio-Rad), band intensity was expressed as the fraction corresponding to GAPDH and analyzed using ImageJ software.

The brains were separated and post fixed in 4% PFA at 4 °C overnight and immersed in 20% sucrose (4% PFA as solvent) followed by 30% sucrose (in 0.1 M PBS). The brain samples were cut into 30-μm-thick sections (CM1850, Leica Microsystems, Wetzlar, Germany). Sections were incubated in 0.01 mol/L citrate buffer, pH 6.0 for high-temperature antigen retrieval. Tissues were blocked in 2% (wt/vol) BSA (Sigma) and then exposed overnight to the following primary antibody mixtures: anti-P2X7R (Abcam, 1:400), anti-GFAP (Thermo, 1:1000), or anti-Iba-1 (Wako, 1:1000) at 4 °C. Detection of primary antibodies was performed with secondary antibodies (donkey-anti-mouse, Alexa 594 conjugated,1:1000, Invitrogen, USA; donkey anti rabbit, Alexa 594 conjugated, 1:1000, Invitrogen, USA; Hoechst, 1: 1000, Beyotime, China) for 1 h in the dark. The sections were then washed five times with PBS in the dark. Immunofluorescent sections were observed with a Leica SP5 fluorescence microscope, using excitation wavelengths of 633 nm (helium/neon2, blue Cy5-labelling), 543 nm (helium/neon1, red Cy3-immunofluorescence), and 488 nm (argon, yellow-green Cy2-immunofluorescence), and images were captured with a CCD spot camera for data analysis. Three regions per hippocampus section and three sections per animal were counted by experimenters who were blind to the experiment design.