Hrp conjugated goat anti rabbit

Total proteins of ischemic brain tissue in each group were extracted by RIPA buffer containing EDTA-free protease inhibitor and phosphate inhibitor (Rocher, Switzerland). After measuring the protein concentration, 30 μg of each sample was subjected to electrophoresis on 15% SDS-PAGE gel at 200 V. The protein was transferred into the PVDF membrane at 100 V in Bio-Rad TransBlot apparatus. 5% skimmed milk diluted by TBST solution was used to block PVDF membranes containing proteins for 2.5 hours at room temperature, and then, the primary antibody, rabbit anti-GADPH (1 : 1000, Abcam, ab181602), NLRP3 (1 : 1000, Abcam, ab181602), caspase 1 (1 : 1000, Abcam, ab181602), and NF-κB (1 : 1000, Abcam, ab181602), were used to incubate PVDF membranes at 4°C overnight. And then, the second antibody HRP-conjugated goat anti-rabbit (1 : 5000; EarthOx, USA) was used to incubate membranes for 2.5 hours after washing three times per 5 mins with TBST solution. Finally, after incubating with enhanced chemiluminescence, the membranes were exposed in ChemiDoc Touch Imaging System. ImageJ software was applied to the quantification analysis of protein bands. Each experiment was performed three times [16 (link)].

UA was purchased from Aladdin (Shanghai, China). Pluronic F127 was purchased from Sigma-Aldrich (St. Louis, MO, USA). C₁₈-PEI was synthesized from our previous methods.^{22 (link)} 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) were obtained from Solarbio Science & Technology (Beijing, China). Annexin V-fluoroisothio cyanate (FITC)/propidium iodide (PI) apoptosis detection kit, Cell Cycle and Apoptosis Analysis Kit and RIPA lysis buffer and blocking buffer were obtained from Beyotime (Shanghai, China). Rabbit polyclonal antibodies against caspase-3 and caspase-8 were purchased from Cell Signaling Technology (Beverly, MA, USA). Rabbit polyclonal antibodies against Fas and FasL were procured from Biosynthesis Biotechnology (Beijing, China). Rabbit polyclonal antibodies against β-actin were supplied by Proteintech (Wuhan, China). HRP-conjugated goat anti-rabbit was from Earthox (Millbrae, CA, USA). Other chemicals, if not specified, were all commercially available and used as received. The deionized water, with a resistivity of 18.2 MΩ cm⁻¹, was obtained from Milli-Q Gradient System (Millipore, Bedford, MA, USA) and used for all of the experiments.