Primary antibodies against zo 1

Cells were collected and lysed in radioimmunoprecipitation assay (RIPA) buffer (Epizyme, Shanghai, China) with protease inhibitor cocktail (MedChemexpress, Monmouth, NJ, USA) and centrifuged at 12,000 rpm for 10 min at 4°C to remove insoluble cell debris. The soluble protein concentration in the supernatants was measured using the bicinchoninic acid (BCA) protein assay kit (NCM Biotech, China) and applied to western blot analyses. Aliquots from each sample were separated by 12% SDS-PAGE and then transferred to polyvinylidene difluoride membranes. The blots were blocked with 5% bovine serum albumin in Tris-buffered saline with Tween-20 at room temperature for 1 h and then incubated overnight at 4°C with primary antibodies against ZO-1, Claudin-5, Occludin (Abcam, Cambridge, MA, USA), VEGFA, and β-actin (Proteintech, Chicago, IL, USA). The blots were subsequently washed and incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Biodragon, Beijing, China) at 37°C for 1 h and visualized with electrochemiluminescence kit (US Everbright, Suzhou, China). Densitometry analysis was analyzed in three independent blots by using Image Lab software version 5.2.1 (Bio-Rad, Hercules, CA, USA).

Pups were sacrificed after prior anesthesia 48 h after HI injury (n=6/group). The cortical tissue in the peri-infarct was dissected out, washed in cold PBS, and homogenized below 4 ℃. The total protein was lysed using radioimmunoprecipitation assay (RIPA) lysis buffer. Then, equal amounts of proteins (30 μg) were separated on 10% SDS-polyacrylamide gels (Bio-Rad, Hercules, CA, USA) and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore Corporation, Billerica, USA). After blocking with 5% nonfat dry milk for 2 h, the membranes were incubated overnight at 4 ℃ with primary antibodies against ZO-1 (1:1,000, Abcam, Cambridge, MA, USA), occludin (1:1,000, Abcam, Cambridge, MA, USA), NF-kB pp65, and HIF-α (both 1:1,000, CST, Danvers, MA, USA). Next, the blots were incubated for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibodies (dilution 1:10,000, Yi Fei Xue Biotechnology, Nanjing, China) at room temperature. Finally, the bands were visualized on a ChemiDoc™ MP System (Bio-Rad, Hercules, CA, USA). The results were quantified by normalizing the relative intensity to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).