Anti claudin 5 antibody

Fresh-frozen temporal cortex tissues were obtained from AD (n = 3) and control (n = 3) individuals. Tissue was cryopreserved in OCT (optimal cutting temperature) compound (Sakura Finetek, Torrance, CA, USA), and 50-μm sections were cut, using a cryostat (Leica, Nussloch, Germany). Slices were fixed with paraformaldehyde, at room temperature, for 20 min, and permeabilized with 100% methanol, at −20 °C, for 20 min. From this step, a brain-slice chamber system maintained the brain-slice preparations in a constantly humid environment. Non-specific sites were blocked with 1% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA), in PBS, for 1 h, at room temperature. Lipofuscin autofluorescence was removed with Sudan B Black (SBB) solution (0.1% SBB in 70% ethanol) for 10 min. Tissues were incubated with the rabbit polyclonal anti claudin-5 antibody (1:300; Abcam; Cambridge, UK), overnight, at 4 °C, and then with FITC-tagged goat–anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 1 h, at room temperature. Sections were counterstained with TO-PRO^®-3 or DAPI. Slices were mounted in Vectashield antifade mounting medium (Vector Laboratories).

Western blot was performed as described previously (7 (link)). Briefly, cellular proteins were prepared in ice-cold RIPA buffer containing protease inhibitor and quantified with Pierce BCA protein assay kit (23,225; ThermoFisher, Waltham, MA). Proteins were separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose blotting membranes (10,600,003; GE Healthcare, Chicago, IL). The membranes were incubated with appropriate primary antibodies (1:1,000 dilution in 5% bovine serum albumin) followed by the peroxidase-conjugated secondary antibody (1:5,000 dilution in 5% nonfat milk). The signals were quantified using G:Box gel imaging system by Syngene (Bangalore, India). The following primary antibodies were used in the present study: anti–VE-cadherin antibody (1:200 dilution, sc-9,989; Santa Cruz Biotechnology), anti-HSPA12B antibody (1:1,000 dilution, a gift from Dr. Zhihua Han [ETSU, Johnson City, TN]), anti-claudin 5 antibody (1:1,000 dilution, ab131259; Abcam, Cambridge, United Kingdom), anti–ZO-1 antibody (1:1,000 dilution, 13,663 s; Cell Signaling Technology, Danvers, MA), anti–β-actin antibody (1:1,000 dilution; 3,700 s; Cell Signaling Technology), and anti-GAPDH antibody (1:1,000 dilution, Cell Signaling Technology, 2,118 s).