Mouse anti integrin β3

To examine the expression of integrin α_Vβ₃, VEGF and its receptor pFlk-1 in the cortex and hippocampus, western blot assay was performed as described in a previous study (Jiang et al., 2013). Samples from treated mice were resolved using sodium dodecyl sulfate polyacrylamide gradient gels (20 mg protein per lane). Proteins were transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked in 5% non-fat milk and then incubated with polyclonal mouse anti-integrin α_v (1 μg/mL), mouse anti-integrin β₃ (1 μg/mL), rabbit anti-VEGF (1 μg/mL) or rabbit anti-pFlk-1 (1 μg/mL) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. After three washes with Tris-buffered saline containing Tween-20, the membranes were incubated with anti-mouse-horseradish peroxidase (Santa Cruz Biotechnology) or goat anti-rabbit-horseradish peroxidase (Santa Cruz Biotechnology) for 30 minutes at room temperature. The experiment was performed in triplicate, and β-actin was used as an internal control. The optical density values were calculated with Quantity One image analysis software (Bio-Rad).