Assessment of Hippocampal BDNF by Western Blot

To analyze the amount of BDNF protein, Western blots were conducted. Brains were removed at 6–7 weeks of age. Hippocampi were manually separated and homogenized at 4°C in Tris–HCl buffer (20 mM, pH 7.4) containing 10% sucrose. Homogenates were centrifuged at 4°C for 30 min with 14,000 g. Pellets were resuspended in ice-cold Tris-HCl buffer, pH 7.4, containing protease inhibitors (Roche Diagnostics). The total protein concentration of each sample was determined by Bradford protein assay (Ultrospec 3000, Pharmacia Biotech) to ensure that the same total amount of protein (10 μg) was applied to the gel. The gel electrophoresis of proteins was carried out using 15% sodium dodecyl sulfate (SDS) polyacrylamide gels run on a mini-gel apparatus (Pequlab) for 1.5 h at room temperature. The Western blot transfer was conducted in a cold buffer (wet conditions) in a Western blot transfer system (Consort) at 400 V, 300 mA for 1.5 h. Membranes were incubated for 1 h in Tris-buffered-saline (100 mM Tris-HCl; 0.9% NaCl, 1% Tween 20, pH 7.4) containing 5% non-fat milk to block non-specific binding sites. Blots were then incubated overnight at 4°C with a primary monoclonal anti-rabbit antibody (1:1,000 dilution, Epitomics, UK cat. no. 3160-1) that labels mature BDNF (14 kDa). Protein β-actin was used as a loading control, labeled with a monoclonal antibody (1:20,000 Sigma, St. Louis, MO Cat no. A5316, RRID:AB_476743). The immunoreactivity was visualized using horseradish peroxidase (HRP)-coupled goat anti-rabbit secondary antibodies (1:20,000 dilution, GE Healthcare) and an enhanced chemiluminescence (ECL) detection system (GE Healthcare). The immunoblots were subsequently analyzed with ImageJ Software (NIH, Bethesda, MD, United States) to measure the optical density of the bands. BDNF expression was measured in proportion to β-actin in the hippocampus (Figure 1B).