Df6387

The protein was extracted from the hippocampal tissues for western blot analysis. After determining the protein concentration using the BCA method, the proteins were processed in 12% sodium dodecylsulphate polyacrylamide gel electrophoresis. After transferring onto the PVDF membrane, the nonspecific staining was blocked by 5% defat milk. The membrane was incubated with the antibodies, including BDNF (1 : 1000, DF6387, Affinity), c-fos (1 : 1000, ab222699, Abcam), and β-actin (1 : 3000, ab8227, Abcam). Thereafter, the membranes were probed with anti-mouse immunoglobulin (Ig) G or anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h.

Tissues were grated and homogenized with RIPA buffer (Boster, Wuhan, China) at 4°C for 30 min, then centrifuged for 15 min at 4°C. The protein concentrations in supernatants were determined by BCA protein assay kit (Boster, Wuhan, China). The protein samples were separated by 10% sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE) electrophoresis, and were then transferred to poly vinylidene fluoride (PVDF) membranes(Millipore, Bedford, MA, USA). Bands were blocked with 5% BSA dissolved in TBST (0.1%Tween 20 in Tris-buffered saline) for 1 h at room temperature. Relative primary antibodies were incubated at 4°C overnight: rabbit mBDNF (1:500, DF6387, Affinity, Cincinnati, OH, USA), rabbit TrkB (1:1,000, AF6461, Affinity, Cincinnati, OH, USA), rabbit phosphorylated p-TrkB (1:1,000, AF3461, Affinity, Cincinnati, OH, USA), and rabbit GAPDH (1:2,000, AF7021, Affinity, Cincinnati, OH, USA). After warming and washing by TBST, second antibody was incubated on bands for 2 h at room temperature: goat anti-rabbit IgG horseradish peroxidase (1:5,000, Promotor, Wuhan, China). Finally, these protein bands were visualized by enhanced chemiluminescence substrate solutions (Promotor, Wuhan, China) with the ChemiDoc XRS chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA).