Reinforced nitrocellulose membranes

For immunoblotting analysis, brains were homogenized by sonication in ice-cold PBS containing 5 mmol/L EDTA, protease and phosphatase inhibitor cocktails (Sigma-Aldrich, Saint Louis, MO, USA). The protein concentration was determined using a Nanodrop 2000 (Thermo Scientific, Waltham, MA, USA). Sample lysates were mixed with 4X LDS Sample Buffer (NuPage, Invitrogen) and heated (70ºC) for 10 minutes. Total proteins (15  μg/well) were resolved on 4% to 12% Tris-Bis gels (NuPage) or on 3% to 8% Tris-Acetate-gels (ZO-1 only) and transferred onto reinforced nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Membranes were blocked in 30 mmol/L Tris–HCl (pH 7.5), 100 mmol/L NaCl and 0.1% Tween-20 (TBS-T) containing 5% fat-free milk powder for 1 hour and then incubated with the following primary antibodies overnight: mouse-anti claudin-5 (1:500), rabbit anti-occludin (1:200), rabbit anti-ZO-1 (1:500), and rabbit anti-β-actin (1:10,000; Sigma-Aldrich). After washing, the membranes were incubated with the appropriate peroxidase-labeled secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 1 hour and visualized using a Super Signal Western Dura substrate (Pierce, Rockford, IL, USA) and a LAS 1000-cooled CCD camera (Fujifilm, Tokyo, Japan). Immunoreactive bands were quantified using the Image Gauge software (Fujifilm) with β-actin used as a loading control.