In the same manner, as described below, western blotting for the determination of BDNF and TrkB was conducted (Park et al., 2019 (link)). Hippocampal tissues were homogenized on ice and lysed in a lysis buffer consisting of 1-mM phenylmethylsulfonyl fluoride, 150-mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholic acid, 1% Nonidet P40, 100-mg/mL leupeptin, 50-mM Tris-HCl (pH, 7.5). Colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA) was used to measure protein content. Total protein 30 μg was separated on SDS-poly-acrylamide gels and transferred to a nitrocellulose membrane, the membrane was blocked with dehydrated milk and then treated with primary antibodies. Mouse anti-β actin (1:3,000; Santa Cruz Biotechnology, CA, USA), rabbit anti-BDNF (1:1,000; Santa Cruz Biotechnology), and rabbit anti-TrkB (1:1,000; Santa Cruz Biotechnology) were used as the primary antibody.
After washing, appropriate horseradish peroxidase-conjugated secondary antibodies were used and incubation was performed at room temperature. Enhanced chemiluminescence detection system (Santa Cruz Biotechnology) was used to measure the expression of bands, and bands were quantified using an Image-Pro Plus computer-assisted image analysis system (Media Cyberbetics Inc., Silver Spring, MD, USA).
After washing, appropriate horseradish peroxidase-conjugated secondary antibodies were used and incubation was performed at room temperature. Enhanced chemiluminescence detection system (Santa Cruz Biotechnology) was used to measure the expression of bands, and bands were quantified using an Image-Pro Plus computer-assisted image analysis system (Media Cyberbetics Inc., Silver Spring, MD, USA).
