2.10. BDNF, TrkB, CREB, and pCREB Expression Measurement

Samples of hippocampus were homogenized on ice in PBS containing 1% protease inhibitor and 1% phosphatase inhibitor for Western-blotting. Proteins were obtained by centrifugation at 14000 rpm at 4°C for 15 min and quantified by Bradford assay (BioRad, USA). A 50 μg sample of each group was subjected to electrophoresis using 20% SDS at 80 V. The proteins were transferred to polyvinylidene fluoride membranes at 250 mA for 2 h. Antibodies for BDNF (1 : 1000, Santa Cruz Biotechnology, USA, number sc-546), TrkB (1 : 1000, Santa Cruz Biotechnology, USA, number sc-8316), CREB (1 : 2000, Cell Signaling Technology, USA, number 9197), pCREB (1 : 2000, Cell Signaling Technology, USA, number 9198), and GAPDH (1 : 10000, KangChen Bio-Tech, Shanghai, China) were applied overnight. Then membranes were incubated with horseradish peroxidase (HRP) secondary antibody (KangChen Bio-Tech, Shanghai, China). At last, the signal intensities of proteins were analyzed using Image J software.