Mouse anti claudin 5 antibody

Immunofluorescence staining was carried out to identify BMECs and to observe the changes in peri-cellular tight junctions and claudin-5 expression. After the BMECs were fixed in 4% paraformaldehyde and then permeabilized with 0.5% Triton X-100 (Sigma) for 15 min, the BMECs were blocked with 5% BSA for 30 min followed by incubation with primary antibodies: rabbit anti-von Willebrand factor (vWF) antibody (1:100, Catalog No. ab6994, Abcam) and mouse anti-claudin-5 antibody (1:50, Invitrogen) at 4 °C overnight. The appropriate secondary antibodies, Alexa Fluor 488-conjugated goat-anti-rabbit or goat-anti-mouse (1:1000, Life Technologies), were then incubated with BMECs at room temperature for 60 min. Nuclei were stained with DAPI (1:1000, Sigma) for 10 min. Cells were observed under a ZEISS LSM 780 confocal microscope (Carl Zeiss, Germany).

For imaging studies, cells or beads were fixed in 4% paraformaldehyde (PFA) or 100% ice-cold methanol followed by permeabilization in 0.25% Triton X-100, washed in PBS, incubated with primary antibody for 1 h, washed in PBS, and then incubated with Alexa Fluor-conjugated secondary antibodies for 30 min. Alexa Fluor 594-conjugated phalloidin and Alexa Fluor 488-conjugated phalloidin were used for actin localization and were purchased from Invitrogen. Mouse anti-Na/K ATPase was purchased from Millipore, mouse anti-VE-cadherin was purchased from R&D Systems, and rabbit anti-ZO-3 antibody and mouse anti-claudin-5 antibody were purchased from Invitrogen. Mouse anti-glial fibrillary acidic protein (anti-GFAP) antibody was purchased from BD Biosciences. Slides/beads were mounted with Vectashield (Vector Laboratories) containing 4′,6-diamidino-2-phenylindole (DAPI). Images were captured on an Olympus FV1000 confocal microscope or an IX83 inverted fluorescence microscope and were analyzed using ImageJ. For scanning electron microscopy (SEM), beads were fixed, processed, and imaged as described previously (42 (link)).

Cells were washed three times with sterile PBS, and cell lysates were obtained using radioimmunoprecipitation assay (RIPA) buffer (ThermoFisher Scientific) plus protease inhibitor cocktail (Roche). The proteins in the lysates were separated by SDS-PAGE on a 5–20% polyacrylamide gel (Wako) and were then transferred to polyvinylidene fluoride membranes (Merk-Millipore). After blocking with 5% skim milk in TBS containing 0.1% Tween 20 at room temperature for 1 h, the membranes were incubated with a mouse anti-claudin-5 antibody (diluted 1/250; ThermoFisher Scientific), a mouse anti-P-gp antibody (diluted 1/250; GeneTex), a rabbit anti-CD31 antibody (diluted 1/1000; abcam), or a mouse anti-ß-actin antibody (diluted 1/5000; Sigma-Aldrich) at 4 ^oC overnight, and then with a HRP-conjugated anti-mouse IgG (Cell Signaling Technology) or a HRP-conjugated anti-rabbit IgG (Cell Signaling Technology) at room temperature for 1 h. The bands were detected with ECL Plus western blotting detection reagents (ThermoFisher Scientific), and the signals were visualized with a LAS-4000 imaging system (Fuji Film).