Western Blot Analysis of Ischemic Brain Tissue

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)].