Rabbit anti zo 1

To determine the change in protein levels in the CC, CC tissue was precisely isolated from the right hemisphere closed to 1.0 mm anterior to the bregma on ice. Once weighed, the tissue was digested in RIPA lysis buffer and homogenized. The protein concentration was quantified, and then the protein were separated on 10% SDS–PAGE gels and transferred to nitrocellulose membranes (Invitrogen, USA). After three times washes in TBS with 0.05% Tween-20 (TBST), the membranes were blocked in TBST with 5% skim milk for 2 h at room temperature. The membranes were incubated with primary antibodies at 4 ℃ overnight and then further incubated with HRP-conjugated secondary antibodies (1:2000) for 1 h at room temperature. The following primary antibodies were used: mouse anti-PDGFR-β (1:1000, Abcam); rabbit anti-TGF-β1 (1:2000, Abcam); rabbit anti-pSmad2 (1:1000, Millipore); rabbit anti-occludin (1:2000, Thermo Fisher); rabbit anti-claudin 5 (1:2000, Thermo Fisher); rabbit anti-ZO-1 (1:1000, Thermo Fisher) and rabbit anti-MBP (1:2000, Abcam). Western Bright ECL solution was used to develop the blots, which were analyzed using GelPro Analyzer 6.0 software (Media Cybernetics, Rockville, MD, USA).

Rabbit anti-p-Akt (Ser473), anti-p-Akt (Thr308), anti-Akt, anti-PDK1, anti-p-PDK1, anti-PI3K p85, anti-p-PI3K p85, and anti-ERK1/2 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse anti-CLDN2, rabbit anti-CLDN2, mouse anti-ZO-1, and rabbit anti-ZO-1 antibodies were from Thermo Fisher Scientific (Rockford, IL, USA). Mouse anti-p-Stat3 (Y705) and anti-Stat3 antibodies were from BD Biosciences (Franklin Lakes, NJ, USA). Goat anti-β-actin and rabbit anti-p-ERK1/2 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fisetin, GEF, and LY-294002 were obtained from Cayman Chemical (Ann Arbor, MI, USA) and dissolved in dimethyl sulfoxide (DMSO). Control cells were treated with DMSO as a vehicle. The concentration of DMSO in the control and drug-treated cells was 0.1%. CDDP and DXR were from Fujifilm Wako Pure Chemical Industries (Osaka, Japan). DOC and hypoxia probe solution (LOX-1) were from Tokyo Chemical Industry (Tokyo, Japan) and Medical and Biological Laboratory (Tokyo, Japan), respectively. All other reagents were of the highest purity available.

Cells were fixed with 2% paraformaldehyde for 20 min at room temperature, followed by blocking with 0.1% BSA, 0.3% Triton X-100, 5% normal goat serum, in 1× PBS. Incubation with the primary antibodies was performed in blocking buffer and done overnight at 4 °C. The working solutions were as follows: rabbit anti-RLBP1 1:200 (PA5–29759, Thermo Fisher, Waltham, USA), rabbit anti-MITF 1:200 (PA5–38294, Thermo Fisher, Waltham, USA), rabbit anti-ZO1 1:100 (61–7300, Thermo Fisher, Waltham, USA), rabbit anti-BEST1 1:100 (ab14928, Abcam, Cambridge, UK). The immunoreactivity of the antibodies was confirmed by immunostainings on human retinal cryosections and ARPE19 cells as positive control (Fig. S1). As a secondary antibody we used the Alexa Fluor 594 goat-anti-rabbit 1:1000 (A-111012, Thermo Fisher, Waltham, USA). Cell nuclei were counterstained with DAPI (Thermo Fisher, Waltham, USA). Cells were imaged using a Leica TCS SP8 X confocal microscope.

Brain sections were fixed in 4% PFA for 15 min. After extensive washes with PBS, the sections were incubated in blocking buffer (1 % BSA in PBS containing 0.3% normal donkey serum and 0.3 % Triton X-100) for 1 h at room temperature. Next, the sections were incubated with primary antibodies [rat-anti-CD31 (1:200, BD Biosciences, 553370), mouse anti-claudin-5 (1:200, Invitrogen, USA, 35-2500), rabbit anti-ZO-1 (1:400, Thermofisher, USA, 61-7300), rabbit anti-caveolin-1 (1:500, Cell Signaling, 3238S), rabbit anti-PDGFRβ (1:200, Cell Signaling, 3169), rabbit anti-AQP4 (1:500, Millipore, USA, AB3594), rat anti-Ly6G (1:200; Biolegend, USA, 108402), rat anti-CD3 (1:200, eBioscience, USA, 14–0032-82), rat anti-CD11b (1:200, BD Biosciences, 553309), mouse anti-glial fibrillary acidic protein (GFAP, 1:200, BD Bioscience, USA, 556327), and rabbit anti-Iba1 (1:500; Wako Inc, USA, 019-19741)] overnight at 4 °C. After extensive washes, the sections were incubated with appropriate fluorescent secondary antibodies. After extensive washes, the sections were mounted with fluoromount-G with DAPI. Images were taken from peri-hematoma regions using a Nikon Eclipse Ti microscope or LSM710 confocal microscope.

Cells were lysed with RIPA buffer (50-mM Tris pH 7.4, 1% NP-40, 0.5% Na-deoxycholate, 1% SDS, 150-mM NaCl, 2-mM EDTA, 1 × protease inhibitor cocktail, and 1 × phosphatase inhibitor cocktail). Total protein levels were determined using the Bio-Rad protein assay kit, and equal amounts of proteins were loaded and separated on SDS-PAGE. After transferring to PVDF membrane (Millipore), proteins were detected using a standard immune-blotting technique. The following primary antibodies were used: mouse anti-claudin-5 (1:500, Invitrogen, USA, 35-2500), rabbit anti-ZO-1 (1:500, Thermofisher, USA, 61-7300), rabbit anti-caveolin-1 (1:1000, cell signaling, 3238S), and mouse anti-β-actin (Sigma, A5441, 1:2000). Target proteins were visualized using SuperSignal West Pico Plus Chemiluminescent Substrate (Thermo scientific). The density of target protein bands was quantified using NIH ImageJ software. The expression of target proteins was normalized to that of β-actin.

The following antibodies were used for western blotting: rat anti-HA (3F10; Roche/Sigma-Aldrich), rabbit anti-myc, mouse anti-RhoA, rabbit anti-MLC2 (Santa Cruz Technology), rabbit anti-FAM40B (Sigma-Aldrich), rabbit anti-pThr18/pSer19-MLC2 (Cell Signaling), mouse anti-GADPH (Millipore), secondary HRP-conjugated sheep anti-mouse IgG and donkey anti-rabbit IgG (GE Healthcare). Antibodies for immunofluorescence analysis were: rabbit anti-HA (Santa Cruz Technology), rabbit anti-pSer19-MLC2 (Cell Signaling), mouse anti-VE-cadherin (BD Biosciences), rabbit anti-ZO-1 (#61–7300; ThermoFisher Scientific), secondary AlexaFluor-488 and -647-conjugated antibodies (Molecular Probes). Cells were also stained with DAPI (DNA) and AlexaFluor-546-labeled phalloidin (F-actin; Molecular Probes).