Anti claudin 5

Mice were sacrificed by cervical dislocation; then, the cortex, the striatum, the cerebellum, and the hippocampus were dissected out. Tissues were homogenized in a lysis buffer containing 20 mM Tris, pH 7.4, 1% Nonidet P-40, 1 mM EDTA, 20 mM NaF, and 2 mM Na3VO4 and a 1:1000 protease inhibitor mixture (Sigma-Aldrich) and sonicated with 2 × 10 s pulses. For claudin-5 and occludin, 20 µg of total protein lysate was immunoblotted with the following antibodies: anti-claudin-5 (1:1000) (Abcam, Cambridge, UK; Cat. N. ab15106) and anti-occludin (1:1000) (ThermoFisher Scientific, Waltham, MA, USA; Cat. N. 710192). For protein normalization, anti-actin (1: 5000) (Sigma-Aldrich; Cat. N. A5441) was used. Protein bands were detected by ECL Prime (GE Healthcare, Chicago, IL, USA) and quantitated with Quantity One Software (Bio-Rad Laboratories, Hercules, CA, USA).

The ipsilateral/right cortex was isolated and homogenized for Western blotting as previously described [15 (link)] with the following primary antibodies: anti-MST1 (1 : 1000, Cell Signaling Technology, Danvers, MA, USA), anti-phosphorylated MST1 (1 : 1000, Cell Signaling Technology, Danvers, MA, USA), anti-ZO-1 (1 : 500 Invitrogen, Grand Island, NY, USA), anti-occludin (1 : 1000 Abcam, Shanghai, China), anti-claudin-5 (1 : 1000, Abcam, Shanghai, China), anti-NF-κB (1 : 1000, Cell Signaling Technology, Danvers, MA, USA), and anti-matrix metallopeptidase- (MMP-) 9 (1 : 1000, Cell Signaling Technology, Danvers, MA, USA). GAPDH (1 : 10,000, Proteintech, Rosemont, IL, USA) was used as an internal loading control. The relative density of the Western blots was normalized to the sham group.

After the experiments, colonic tissues were dissected and fixed with 4% paraformaldehyde. Paraffin sections of colonic tissues were dewaxed and hydrated. The sections were incubated in QuickBlock™ Blocking Buffer (Beyotime, Shanghai, China) for 30 minutes at room temperature. Then, the sections were incubated with the anti-zonula occludens 1 (ZO-1; Abcam, Cambridge, MA, USA; ab221547; 1/100), anti-claudin-5 (Abcam, Cambridge, MA, USA; ab4648; 1/100), or anti-NF-κB p65 (Cell Signaling Technology, Danvers, MA, USA; 8242; 1/400) at 4°C overnight and washed 3 times with phosphate-buffered saline (PBS). The staining of colonic tissues was observed under a fluorescence microscope BX53 (Olympus, Tokyo, Japan) at 100x or 200 magnification.

To understand changes at the protein level, whole cell lysates prepared in radioimmunoprecipitation assay (RIPA) buffer were subjected to SDS-PAGE and western blotting, as described elsewhere. The following antibodies were used in this study: anti-claudin-5, anti-ADAM17 (Abcam, Cambridge, UK), anti-CD144, anti-β-catenin (Genetex, Irvine, CA), anti-occludin (Proteintech, Chicago, IL), anti-ZO-1 (Invitrogen, Waltham, MA), and anti-β-actin (Sigma-Aldrich, Chicago, IL). Densitometry of protein bands was performed using the Gel-Pro Analyzer software (Media Cybernetics, Rockville, MD, USA). β-actin was used as the loading control.

The lower right lung lobes were removed and stored in liquid nitrogen for protein analysis. Briefly, the lung tissues were lysed on ice in a RIPA (P0013B, Beyotime) buffer containing 1 mM PMSF (Beyotime). After sonication, the protein concentration in the supernatants was measured using a bicinchoninic acid protein assay kit (Beyotime). Equal amounts of protein were loaded in 10% SDS–PAGE, and then the proteins were transferred to polyvinylidene fluoride membranes and blocked in 5% bovine serum albumin for 1 h at room temperature. After rinsing in PBST 5 times, the membranes were incubated with each primary antibody overnight at 4°C: the anti-claudin 5 (Abcam, ab15106), the phosphor-MAPK family Antibody sampler kit (cell-signaling technology, #9910) the MAPK family Antibody sampler kit (cell-signaling technology, #9926), and β-actin (cell-signaling technology, #4970). After washing with PBST, the membranes were finally incubated with the appropriate secondary antibody for 1 h at room temperature. Protein bands were detected using an ECL High-Signal reagent (Thermo). The band intensity was quantified using the ImageJ software.

The mice were sacrificed by cardiac perfusion with 0.9% saline, and their brains were cut into 20 μm coronal sections after being fixed in 4% paraformaldehyde. The brain sections were incubated with anti‐MBP (1:400, Abcam), anti‐SMI32 (1:400, Covance), anti‐CD31 (1:200, Santacruz), anti‐claudin‐5 (1:400, Abcam), anti‐occludin (1:400, Invitrogen), anti‐GFAP (1:400, CST), anti‐MPO (1:400, CST), anti‐MMP‐9 (1:400, CST) antibodies, respectively, overnight at 4°C. After being washed with PBS, the sections were incubated with the appropriate secondary antibodies for 2 h in the dark at room temperature. The sections were imaged using an Olympus microscope with an IX71 digital camera (Olympus), and the images were quantified with IPP 6.0 software. All evaluations were performed by a blinded investigator.