Rabbit anti β actin

Total proteins were extracted from the hippocampus tissues, and protein concentration was assessed by the bicinchoninic acid (BCA) reagent (Thermo Fisher, Waltham, MA, USA) method. Equal amounts of protein were subjected to SDS-polyacrylamide gel electrophoresis. Separated proteins were transferred from the gel onto PVDF membranes (Bio-Rad, Hercules, CA, USA) by a transfer apparatus. The membrane was blocked with 5% fat-free dry milk for 2 h at room temperature. Subsequently, blots were incubated overnight at 4°C with indicated primary antibodies, including rabbit anti-caspase-3 (Sigma, St. Louis, MO, USA, 1 : 1000), rabbit anti-Bax (Sigma, 1 : 1000), rabbit anti-Bcl-2 (Sigma, 1 : 500) and rabbit anti-β-actin (Sigma, 1 : 1000). The membranes were then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Sigma, 1 : 5000) for 2 h at 37°C. Finally, immunoreactive bands of the protein expression level were normalized to the intensity of the corresponding bands for β-actin. Densitometric analysis of the results was performed with Image J 1.41 software (National Institutes of Health, Bethesda, MD, USA).

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.