Protein Expression Analysis of Ischemic Penumbra

Total protein was extracted from the penumbral region of ischemic hemispheres, different conditions-treated ECs or BV₂ microglia as described previously (Wang et al., 2016 (link)). Then, 20 μg of protein samples were subjected to 10–15% Bis-Tris gel electrophoresis and transferred onto a 0.2 μm polyvinylidene fluoride membranes (Millipore Corporation, United States). The membranes were blocked with 5% non-fat dry milk-TBS-0.1% Tween 20 for 2 h and washed. Then, the membranes were incubated with primary antibodies against TLR4, MyD88, p-p65, p65 (1:200, all from Santa Cruz, CA, United States), PPARγ, NLRP3, caspase-1, lba1, CD68 (1:1000, both from Abcam, United States), occludin, ZO-1 and β-actin (1:1000, all from Cell Signaling Technology, United States), overnight at 4°C. This was followed by incubation with appropriate horseradish conjugated secondary antibodies for 2 h. Immunoreactivity was detected by the BeyoECL Plus kit (Beyotime, China) on an image system (ChmiScope 2850; Clinx Science Instruments, China); band intensities were analyzed and calculated.