Hippocampal BDNF and TrkB Expression Analysis

Western blotting for BDNF and TrkB expressions was performed, according to the previously described method (Kim et al., 2014 (link)). The hippocampal tissues were collected, and then were immediately frozen at −70°C. The hippocampal tissues were homogenized on ice, and lysed in a lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 1 mM EGTA, 1.5 mM MgCl₂ 6H₂O, 1 mM sodium orthovanadate, and 100 mM sodium flouride. Protein content was measured using a Bio-Rad colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA). Protein (30 μg) was separated on SDS-polyacrylamide gels and transferred onto a nitro-cellulose membrane. Mouse actin antibody (1:1,000; Santa Cruz Biotechnology), rabbit BDNF antibody (1:1,000; Santa Cruz Biotechnology) and rabbit TrkB antibody (1:1,000; Santa Cruz Biotechology) were used as the primary antibodies. Horseradish peroxidase-conjugated anti-rabbit antibody for BDNF (1:2,000; Vector Laboratories) and TrkB (1:3,000; Vector Laboratories) were used as the secondary antibodies. Band detection was performed using as enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech GmbH, Freiburg, Germany).