2.5. Western Blotting Analysis

After 24 h reperfusion, the rats were deeply anesthetized, and their brains were quickly removed following cardiac perfusion with 200 ml normal saline. The levels of pyroptosis-related proteins and Aβ_1-42 oligomers were detected by Western blotting. In brief, after concentrations measurement and protein denaturation, equal amounts of protein samples extracted from ischemic penumbra and equivalent area under sham were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto the polyvinylidnene fluoride membranes (Millipore, Billerica, MA, USA). Subsequently, the membranes were blocked at room temperature with 5% bovine serum albumin (BSA) for 1 h and incubated with the following primary antibodies at 4°C overnight: anti-GSDMD, anti-β-actin (CST, Danvers, MA, USA), anticaspase-11, anti-IL-6, anti-IL-1β (Santa Cruz, Dallas, TX, USA), anti-NLRP3, anticaspase-1 (Proteintech, Rosemont, IL, USA), and anti-Aβ_1–42 (Abcam, Cambridge, UK). Then, the membranes were washed and incubated with corresponding secondary antibody (SAB, College Park, MD, USA) for 1 h at room temperature. After developing by the enhanced chemiluminescence kit (Millipore), pictures were captured with a gel imaging instrument (BioRad Laboratories, USA), and the intensities were analyzed by ImageJ software (National Institutes of Health, USA).