Horseradish peroxidase conjugated anti rabbit secondary antibody

Ipsilateral brain tissue samples were homogenized in radioimmunoprecipitation assay lysis buffer, and centrifuged at 12,000 r/min for 5 minutes at 4°C. Supernatants were collected. Protein concentration was determined by bicinchoninic acid assay. Equal amounts of protein (30 μg per lane) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membranes. Membranes were incubated with rabbit anti-synaptophysin (1:500; Proteintech), rabbit anti-PSD 95 (1:1000; Cell Signaling), and rabbit anti-synapsin I (1:1000; Cell Signaling) overnight at 4°C. After washing, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:5000; Proteintech) for 50 minutes at room temperature. Enhanced chemiluminescence solution (Thermo Fisher Scientific) was used for detecting immunoreactive bands. Last, membranes were developed using a Bio-Rad ChemiDoc XRS digital documentation system (Bio-Rad). Synapsin band densities were quantified and normalized using mouse anti-β-actin (1:4000). Results are presented by averaging the values (protein/β-actin).