Ab59720

Mice were anesthetized i.p. with 4% chloral hydrate (0.1 mL/10 g body weight) and transcardially perfused with 100 mL normal saline, followed by 100 mL of 0.1 M PBS containing 4% PFA (pH 7.4). Brains were post-fixed in 0.1 M PBS + 4% PFA overnight and subsequently dehydrated in 40% sucrose solution. Coronal brain sections (20 µm thick) were obtained using a cryostat (CM1950; Leica Microsystems, Wetzlar, Germany) and blocked for 1.5 h in 5% bovine serum albumin (BSA) in PBS with 0.1% Triton X-100. Sections were incubated overnight at 4°C in 3% BSA in PBS containing 0.1% Triton X-100 with an anti-ZO-1 antibody (1:50; ab59720, Abcam). After rinsing three times with PBS, sections were incubated for 2 h with a fluorochrome-coupled secondary antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime Institute of Biotechnology, Shanghai, China). After rinsing with PBS, sections were visualized under a fluorescence microscope (Leica Microsystems).

The rats (n = 5 in each group) were anesthetized with 3% pentobar bital sodium (50 mg/kg), and then, the rats were decapitated, and the brain was removed and stored in 4% paraformaldehyde until processing. Frozen areas (10 μm) of the injured cortex were brought to room temperature and were then incubated in 3% H₂O₂ for 10 min. After washing three times in phosphate buffer saline (PBS) for 5 min each at room temperature, 5% normal donkey serum was used to block the nonspecific binding. Immunostaining was performed using primary antibodies (1:400, Abcam ab59720, USA) specific for ZO-1 and antibodies for MMP-9 (1:400, Epitomics 2551-1, USA) at 4°C overnight. Next, staining with biotin-labeled secondary antibodies for 120 min was performed. Then, the avidin-biotin-peroxidase complex (1:100, Sigma, USA) was used at 37°C for 1 h. Diaminobenzidine (Boster Biotech Co. Wuhan, China) was applied to visualize the immunoreactivity. Under the light microscope, positive staining (brown yellow) was located for ZO-1 and MMP-9. The images were under a magnification of 400 × by randomly choosing 10 microscopic fields from each group, and the digital software Image-ProPlus 5.0 (Media Cybernetics, USA) was used to automatically detect the integral optical density of each group.

Sections (colon section, 5 μm; brain serial sagittal section, 30 μm) were treated for 20 min with 0.6% H₂O₂ in PBS, permeabilised in PBS-Triton X-100 0.25% (PBS-Tx) for 30 min and blocked for 1 hr with 10% fetal bovine serum (FBS) in PBST×0.1%. TNFα (1:500 dilution, Abcam, ab34674) and ZO-1 (1:500 dilution, Abcam, ab59720) were incubated overnight at 4°C, followed by secondary antibodies (1:500 dilution) for 1hr at room temperature. Positive signals were visualized using 3,3-diaminobenzidine (DAB) (Sigma-Aldrich, Saint Louis, MO, USA). Images were acquired with DM2500 microscope (Leica, Milano, Italy) equipped with DFC350FX digital camera (Leica).