Goat anti mouse igg h l

Total proteins were extracted from the right cerebral cortex in the ischemic penumbra and quantitated using a BCA kit (Beyotime, Shanghai, China). Equal amounts of protein were separated with 8–12% SDS-PAGE gels and electrically transferred to PVDF membranes (Millipore, Burlington, MA, USA). The membranes were incubated with primary antibodies against Bcl-2 (1:1000, T40056, Abmart, Shanghai, China), Beclin1 (1:2000, ab207612, Abcam, Cambridge, UK), Bax (1:10,000, 50599-2-Ig, Proteintech, Wuhan, China), caspase3 (1:1000, T40044, Abmart, Shanghai, China), LC3B (1:2000, T40044, Abcam, Cambridge, UK), ZO-1(1:2000, 21773-1-AP, Proteintech, Wuhan, China), Occludin (1:10,000, 66378-1-Ig, Proteintech, Wuhan, China), β-actin (1:100,000, AC026, Abclonal, Wuhan, China), and GAPDH (1:10,000, 60004-1-Ig, Proteintech, Wuhan, China) at 4 °C overnight. They were then incubated in Peroxidase-Conjugated Goat Anti-Rabbit IgG(H+L) or Goat Anti-Mouse IgG(H+L) (Yeasen, Shanghai, China) diluted at 1:10,000 for 1 h at room temperature. After chemiluminescence detection had been performed using ECL reagents (Yeasen, Shanghai, China) and a Fusion Edge Multi-function Imaging System (Vilber, Collégien, France), the relative optical densities of the protein bands were analyzed using ImageJ software.

Aortic sinus sections were rewarmed for 10 min at room temperature. Tissues fixation with 4% paraformaldehyde for 15 min, non−specific antigen blocking with 5% BSA in PBST for 10 min, NF-κB primary antibody (1:200 dilution) incubation at 4°C overnight, goat anti-mouse IgG H&L (1:200 dilution) linking for 1 h and 4’,6-diamidino-2-phenylindole (DAPI) (Yeasen, China) counterstaining were performed. The images were captured using a fluorescence microscope and merged.