Las4000 ccd imaging system

Whole cell lysate extracts were prepared by lysing hemibrains in lysis buffer (25 mM Tris, pH 7.5, 0.15 M NaCl, 1 mM phenylmethylsulfonylfluoride, 1% Triton X-100 and complete protease and phosphatase inhibitor cocktails, Sigma-Aldrich, Inc.; St. Louis, MO, USA) using a beadbeater (2 pulses for 30 s each at 4500 rpm). Cell debris was removed by centrifugation at 14,000 rpm for 20 min at 4 °C. Amount of protein in each sample was determined using the BCA protein assay kit (Pierce; Rockford, IL, USA). 20 µg of protein lysate was subjected to electrophoresis in denaturing 4–12% SDS-PAGE. Membranes were probed for caveolin-1 (1:1000; Cell Signaling Technology; Beverly, MA, USA), claudin-5 (1:1000; ThermoFisher Scientific; Waltham, MA, USA), claudin-12 (1:1000; ThermoFisher Scientific; Waltham, MA, USA) and ZO-1 (1:1000; Invitrogen; Carlsbad, CA, USA) overnight at 4 °C. As a loading control, membranes were probed for β-actin (1:10,000). The enhanced chemiluminescence western blot was digitalized with a LAS4000 CCD imaging system (GE Healthcare, USA) and analyzed by ImageQuant TL software.

The hemibrain ipsilateral to CCI/sham surgery was homogenized and extracted in Tris-EDTA extraction buffer (20 mM TRIS, 150 mM NaCl, 2 mM EDTA, 2% Triton X-100) with protease inhibitors. The brain extracts were run on a 4–12% Bis-Tris gels and transferred onto nitrocellulose membranes (Invitrogen, USA). The membranes were probed with a AQP-4 (Abcam, USA), HIF-1α (Novus Biologicals, USA), MMP-2 (R&D systems, USA) and MMP-9 (R&D systems, USA) primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, USA). To verify uniformity of protein loading membranes were stripped and reprobed with GAPDH or β-actin antibody (Cell Signaling Technology, USA). The enhanced chemiluminescence western blot was digitalized with a LAS4000 CCD imaging system (GE Healthcare, USA) and analyzed by ImageQuant TL software.