Claudin 5 antibody

To extract protein from HBMECs, cells were washed with 1× PBS and lysed in CelLytic MT Cell Lysis Reagent (MilliporeSigma) supplemented with Protease Inhibitor (Roche, 11836170001). A standard protocol was used for Western blotting. Briefly, samples were resolved in an SDS polyacrylamide gel, blotted onto a 0.45 μm PVDF membrane (Thermo Fisher Scientific), and incubated overnight at 4°C with the following primary antibodies: ASL antibody (Abcam, ab201025), claudin-1 antibody (Thermo Fisher Scientific, 51-9000), claudin-5 antibody (Thermo Fisher Scientific, 35-2500), occludin antibody (Thermo Fisher Scientific, 33-1500), VE-Cadherin antibody (R&D Systems, AF938), and α-Tubulin antibody (MilliporeSigma, T5168). The membranes were incubated with the corresponding secondary antibodies on the following day. Western blot images were taken using the ChemiDoc MP Imaging System (Bio-Rad) and further analyzed using Bio-Rad Image Lab Software.

WT and PECAM-1^−/− pMBMECs were seeded on matrigel-coated 16-well glass Nunc™ Lab-Tek™ chamber slides (Thermo Fisher Scientific) or removable 12-well chamber glass slides (Ibidi). If required, pMBMECs were stimulated at the 6th day after isolation with IL-1β (20 ng/ml) in the presence of serum for 16 h. Seven days after isolation, cells were fixed with 100 μl 1% PFA for 10 min or, in the case of occludin and VE-cadherin, in −20°C-cold methanol for 30 s. Thereafter, cells were permeabilized in blocking buffer (5% skimmed milk, 0.3% Triton X-100, 0.04% NaN₃ in TBS) for 20 min. Hybridoma supernatants [rat-anti-mouse: PECAM-1 (Mec13.3), VE-cadherin (11D4), ICAM-1 (25ZC7), JAM-A (BV12); rat-anti-human CD44 (9B5) used as isotype control] were applied without prior dilution. ZO-1 antibody (rabbit polyclonal; 1:100; Thermo Fisher Scientific), rabbit polyclonal occludin antibody (1.25 μg/ml Thermo Fisher Scientific), rabbit polyclonal claudin-5 antibody (1.25 μg/ml, Thermo Fisher Scientific) and rabbit IgG (R&D Systems) were diluted in blocking solution. Primary antibodies were incubated for 30 min at room temperature. After washing of wells with PBS, secondary antibodies (goat-anti-rat Alexa488, Thermo Fisher Scientific; goat-anti-rabbit Cy3, Jackson ImmunoResearch) were applied together with DAPI (0.5 μg/ml) for 30 min at room temperature and slides were mounted in Mowiol.