Monoclonal rat α zo 1

BREC monolayers were fixed with 1% paraformaldehyde, permeabilized with 0.2% Triton X-100 and blocked with 10% goat serum, followed by their incubation with primary antibodies: polyclonal rabbit α-Claudin-5 (Invitrogen; 1:100), monoclonal mouse α-Occludin (Invitrogen; 1:100), monoclonal rat α-ZO-1 (Millipore; 1:100), or monoclonal mouse α-VE-Cadherin (F-8) (Santa Cruz Biotechnology; 1:100), for 2 days at 4°C. The primary antibodies were detected using secondary fluorescent antibodies: goat anti-mouse Alexa Fluor 488 (Life Technoligies, 1:400), goat anti-rabbit Alexa Fluor 594 (Life Technologies, 1:400), goat anti-rat Alexa Fluor 647 (Life Technologies, 1:400) and Hoechst (Life Technologies, 1:1000) overnight at 4 °C. Samples were imaged using a confocal microscope (TCS SP5; Leica, Wetzlar, Germany).

Total protein content of confluent BREC monolayers was quantified by Western immunoblotting. BREC were harvested in a detergent-based extraction buffer. After the determination of total protein concentrations using DC protein assay kit (Bio-Rad), fifty micrograms protein was diluted in LDS sample buffer and was loaded on 4%–12% Bis-Tris 1.5-mm, 10-well gels (NuPAGE, Invitrogen). The gels were run according to NuPAGE protocols using MOPS SDS running buffer (Invitrogen), transferred to MSI nitrocellulose (Fisher Scientific, Pittsburgh, PA), blocked in 2% ECL Advance blocking reagent (Invitrogen) in 0.5% TBS-T, and immunoblotted with: polyclonal rabbit α-Claudin-5 (Invitrogen; 1:1000), polyclonal rabbit α-Occludin (Invitrogen; 1:1000), monoclonal rat α-ZO-1 (Millipore; 1:1000), or monoclonal mouse α-β-Actin (Cell Signaling; 1:5000). Primary antibodies were detected with horseradish peroxidase-conjugated α-mouse, α-rabbit or α-rat IgG and chemiluminescence (ECL Plus; GE Healthcare, Buckingham-shire, England). Images were acquired with FluorChem™ E chemiluminescent detector (Protein Simple, San Jose, CA) and the intensity of each band was analyzed using ImageJ 1.46r (National Institutes of Health, USA) software.