Western blot assay

Ground brain tissues (20 mg) were added to RIPA lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) containing protease inhibitor, and total proteins were extracted by centrifugation at 14,000 x g for 10 min at 4˚C. The OGD/R cells (1x10⁶/well) were washed once with ice cold PBS and lysed with RIPA lysis buffer on ice for 30 min, after which the total protein was also extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology). An Ultra-Bradford Protein Assay kit (Sangon Biotech Co., Ltd.) was used to determine the concentration of proteins. The proteins (30 µg/lane) were subjected to 10% SDS-PAGE, and then transferred onto a PVDF membrane (Roche Diagnostics). After blocking the PVDF membrane with 5% BSA for 1 h, the membrane was incubated with primary antibodies overnight at 4˚C. The secondary antibody HRP-labeled goat anti-rabbit IgG (dilution 1:5,000; cat. no. ab6721; Abcam) was then applied to react at room temperature for 1 h. After visualization of the bands using an ECL reagent (cat. no. sc-2048; Santa Cruz Biotechnology, Inc.), the results were recorded using a gel imaging system (Kodak film developer; FUJIFILM Wako Pure Chemical Corporation) and analyzed using ImageJ software (version 7.0; National Institutes of Health). The primary antibodies targeted MMP-2 (1:1,000; cat. no. ab97779), MMP-9 (1:1,000; cat. no. ab38898), MMP-8 (1:500; cat. no. ab53017), TIMP-1 (1:1,000; cat. no. ab211926) and GAPDH (1:2,000; cat. no. ab9482), and were all acquired from Abcam.