Horseradish peroxidase conjugated anti rabbit or anti mouse igg

Total protein in hippocampal tissue was extracted with a protein extraction kit (Beyotime), and protein concentration was determined using the Bradford protein concentration determination kit (Beyotime). Total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Afterwards, the separated proteins were transferred onto nitrocellulose filter membranes. The blots were blocked with 5% bovine serum albumin for 2 hours at room temperature, and then incubated with the following primary antibodies at 4°C overnight: rabbit monoclonal anti-BDNF (1:1000; Abcam), rabbit polyclonal anti-p-ERK1/2 (phosphoT202+Y204; 1:1500; Abcam), rabbit polyclonal anti-ERK1/2 (1:1000; Abcam) and mouse monoclonal anti-GAPDH (1:1000; Abcam). The next day, the membranes were washed three times with phosphate-buffered saline containing Tween-20 (PBST), and then incubated with secondary antibody (horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG; 1:1000; Beyotime, Shanghai, China) for 1 hour. The blots were then washed three times with PBST, and then treated with 1–2 mL enhanced chemiluminescence reaction liquid for 1 minute. The membranes were observed and photographed with a Tanon 5200 Luminescence imaging system (Tanon, Shanghai, China). Protein expression was analyzed according to optical density.