Rabbit anti claudin 5

Brain lysates were separated by NuPAGE 4%–12% Bis-Tris gels (Cat# NP0324BOX, Invitrogen) [19 (link)], transferred to PVDF membranes, and analyzed by Western blotting using rabbit anti-claudin-5 (Cat# ab131259, Abcam, Cambridge, UK; 1:1000 dilution in Western Antibody Dilution Buffer), goat anti-MPO heavy chain (Cat# AF3667, R&D; 1:400 dilution in Western Antibody Dilution Buffer), and mouse anti-β-actin (Cat# A4700, Sigma-Aldrich; 1:2000 dilution in Western Antibody Dilution Buffer) antibodies. Immunoreactive bands were detected using SuperSignal West Pico PLUS chemiluminescence substrate (Cat# 34080, Thermo), imaged using a luminescent image analyzer LAS-3000 (Fujifilm), and quantified by NIH ImageJ.

To prepare whole cell and MP lysates from D3 cells cultured in six-well transwell plates, media were removed from the top and bottom chambers and MPs isolated as described. Cells were washed with PBS and lysed in 100 μl of Laemmli buffer (Biorad, Hercules, CA) per insert. Cell lysates were then harvested by cell scraping, lysates were sonicated for 15 s, boiled and frozen at −80°C. For MP western blotting, MP pellets were also lysed in Laemmli buffer, sonicated, boiled, and frozen at −80°C. Membranes were probed with rabbit anti-caveolin-1 [1:1000, Cell Signaling Technologies (CST), Danvers, MA], rabbit anti-β-tubulin (1:1000, CST), rabbit anti-claudin-1 (1:1000, CST), rabbit anti-claudin-3 (1:1000, Abcam, Cambridge, MA), rabbit anti-claudin-5 (1:5000, Abcam), rabbit anti-occludin (1:1000, Abcam), and rabbit anti-VE-cadherin (1:1000, Abcam). Biorad ECL reagents were added to membranes, which were then developed on blue x-ray film (Phenix Research Products, Candler, NC). Densitometry was performed using ImageJ software analysis (NIH).

There were 6 mice conducted western blotting. Brain tissue surrounding the haematoma was homogenized by sonication in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors (Roche Diagnostics) and centrifuged at 12,000g for 20 min. Proteins were resolved by 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. After being blocked with 5% skim milk in Tris-buffered saline with Tween-20 (TBST), the membranes were incubated with primary antibodies (rabbit anti-claudin-5, 1:1000, Abcam; rat anti-ZO-1, 1:1000, Abcam; rabbit anti-SIRT1, 1:1000, Cell Signaling Technology; rabbit anti-phospho-AMPK(Ser 485), 1:1000, Cell Signaling Technology; rabbit anti-p-AMPK, 1:1000, Cell Signaling Technology; mouse anti-β-actin, 1:2000, Zhongshan Jinqiao, Beijing) overnight at 4 °C. Subsequently, the PVDF membranes were washed three times with TBST and incubated with secondary antibodies (1:5000, Zhongshan Jinqiao, Beijing) at room temperature for 60 min. The blots were visualized using enhanced chemiluminescence (ECL, Cell Signaling Technology) reagents, and band intensities were assessed with Image J software.

hCMEC/D3 were grown to confluence on glass coverslips (Thermo Scientific) coated with rat tail collagen type I solution and then washed in PBS and fixed with methanol 15 minutes at RT (for tight junctions) or in PFA 4% 30 minutes in 4 °C (for LRP1). Fixed cells were permeabilized with 0.1% TritonX-100 and blocked with 5% BSA followed by incubation with primary antibodies against claudin-5 (Rabbit anti Claudin5, Abcam, 1:200), occludin (Rabbit anti Occludin, Invitrogen™, 1:50) or LRP1 (Rabbit anti LRP1 antibody, Abcam 1:100). After washing, cells were incubated with Alexa Fluor-488 donkey anti-rabbit IgG antibody (Life technologies, 1:1000). Then, coverslips were mounted with Fluoroshield™ with DAPI (Sigma-Aldrich) and visualized and photographed with Confocal Microscope (Leica SP2 AOBS SE) for tight junctions or by Zeiss Axio Imager Z1 equipped with an Axiocam MR3.0 camera and Axiovision 4.7 software.

Western blot was performed as previously described.¹⁶ The right brain hemispheres were homogenized in RIPA (Santa Cruz Biotechnology) and centrifuged at 14,000 g at 4°C for 30 min. Equal samples were loaded on an SDS‐PAGE gel, and then the proteins were electrophoresed and transferred to a nitrocellulose membrane. The membranes were incubated overnight at 4°C with the primary antibodies as followed: rabbit anti‐TREM‐1 (1:1000, Abcam), rabbit anti‐SYK (1:1000, Abcam), rabbit anti‐β‐Catenin (1:3000, Abcam), rabbit anti‐phospho‐β‐Catenin (1:1000, Cell Signaling Technology), rat anti‐ ZO‐1 (1:500, Santa Cruz Biotechnology), rabbit anti‐Claudin‐5 (1:1000, Abcam), and mouse anti‐β‐actin (1:3000, Santa Cruz Biotechnology). The respective secondary antibody (1:3000, Santa Cruz Biotechnology) were incubated at room temperature for 2 h. Immunoblots were probed with an ECL Plus chemiluminescence reagent kit (Amersham Biosciences) and visualized with an imaging system (Bio‐Rad, VersaDoc, model 4000). Relative density was analyzed using Image J software.

For immunofluorescence analysis, the following primary antibodies were used: rabbit anti‐ZO‐1 (1:100 dilution, Thermo Fisher, #61‐7300), rabbit anti‐claudin‐5 (1:400, Abcam, #ab53765), rabbit anti‐GFAP (1:500, DAKO, #Z0334), rat anti‐CD31 (clone Mec13.3, BD Biosciences), Pals1 (1:100, Merck, #7‐708), rat anti‐Crb3 (14F9, Abcam, ab180835), mouse anti‐E‐cadherin (1:200, BD Bioscience, #610181), and rabbit anti‐cleaved caspase‐3 (Asp175, 1:100 Cell Signaling, #9661). Secondary antibodies were 3 mg/ml Alexa 546‐conjugated goat anti‐rabbit IgG and 3 mg/ml Alexa 488‐conjugated goat anti‐rat IgG (all from Invitrogen). For Western blotting, rabbit anti‐MPDZ (1:500, Thermo Scientific, 42‐2700) and mouse anti‐beta actin (1:2,500, Sigma‐Aldrich #A5441) were used.