20 μm mesh nylon filter

Brain microvessels were isolated according to the procedure described previously [50 (link)]. For each experiment, the brains of two rats were homogenized in ice-cold Hank’s buffered salt solution (HBSS; 14065-056; Gibco, USA) (4 mL per gram of the tissue) using Glas-Col homogenizer by 20 up-and-down strokes at 400 rpm. The homogenate was centrifuged at 1000×g for 10 min. The pellet was resuspended in 15 mL 20% dextran (70 kDa, TCI, Tokyo, Japan) and then centrifuged at 4500×g for 15 min. The resulting pellet was dissolved in 4 mL HBSS containing 1% bovine serum albumin (BSA; Sigma-Aldrich) and the suspension was passed through a 100-μm mesh nylon filter (BD Falcon, Durham, NC, USA) and then a 20-μm mesh nylon filter (Millipore, Temecula, CA, USA). Brain microvessels retained on the filter were collected with 4 mL HBSS containing 2% proteinase inhibitors (Roche Diagnostics, Indianapolis, IN, USA), and centrifuged at 4500×g for 15 min. For immunoblot analysis, the pellet of microvessels was dissolved in capillary lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, and 2% protease inhibitors) on ice. The lysate was centrifuged at 10,000×g for 10 min, and the supernatant was collected, aliquoted, and stored at −70 °C. Protein expression of RAGE, LRP-1, P-gp, ZO-1, and occludin was examined by Western blot analysis.