Image analyzer

Samples were solubilized in lysis buffer [20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride, 1μg/ml leupeptin, and 2 μg/ml aprotinin] for 1 h at 4°C. The lysates were then clarified by centrifugation at 15,000 g for 30 min at 4°C, were diluted in Laemmli sample buffer containing 2% SDS and 5% (v/v) 2-mercaptoethanol, and were heated for 5 min at 90°C. The proteins were separated via SDS polyacrylamide gel electrophoresis (PAGE) using 10% or 15% resolving gels followed by transfer to nitrocellulose membranes (Bio-Rad, Hercules, CA) and then probed with antibodies against HIF-1a (Novus), CD31 (Abcam), HGF (Santa cruz), VEGF (Abcam), and FGF2 (Abcam) for 1h at room temperature (Table 2). Peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG and enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) were used as described by the manufacturer for detection. The membranes were scanned to create chemiluminescent images that were then quantified with an image analyzer (Kodak).

Equal amounts of total protein samples from the hippocampus were separated via SDS/PAGE (10% Bis‐Tris gel; Invitrogen), following which they were transferred onto a PVDF membrane (Bio‐Rad Laboratories) and blocked in TBST buffer (25 mM Tris‐HCl, 160 mM sodium chloride, and 0.05% Tween‐20) containing 5% (wt/vol) bovine serum albumin (Santa Cruz Biotechnology) for 1 hr at 25°C. The membrane was then incubated with primary antibodies overnight at 4°C. This was followed by incubation with the corresponding secondary antibody for 1 hr at room temperature. Primary antibodies included mouse anti–cleaved caspase‐3 (1:400, Cell Signaling Technology), mouse anti‐Bax (1:1000, Santa Cruz Biotechnology), mouse anti‐Bcl‐2 (1:500, Santa Cruz Biotechnology), mouse anti‐p65 (1:500, Santa Cruz Biotechnology), and rabbit anti‐GAPDH (1:600, Cell signaling Technology). Immunoblot bands were visualized using an ECL Western blot kit (NeoBioscience Biotechnology, China). An Eastman Kodak Image Analyzer (Rochester, NY) was used for semiquantification of the protein signal intensity.