Rabbit anti nmdar1

Paraffin sections were immersed in 3% hydrogen peroxide for 15 min in order to eliminate the activity of endogenous peroxidase. After nonspecific antigen blocking in 2% bovine serum albumin (BSA), sections were respectively incubated with rabbit anti-CaMK II (1:200, santa cruz), rabbit anti-NMDAR1 (1:400, Epitomics), rabbit anti-GAP-43 (1:300, Cell Signaling Technology) primary antibody at 4°C overnight, then with biotinylated-conjugated anti-rabbit secondary antibody (1:800,Proteintch) for 2 h at 37°C. Followed with avidin-biotin-peroxidase complex (ABC) (1:100, Vector) for 1 h at 37°C. Immunoreactivity was visualized with diaminobenzidine (DAB, Boster Biotech).

Tissues were homogenized in RIPA lysis buffer with 1 mM phenylmethyl sulfonyl fluoride (PMSF) and 1% inhibitor cocktail (Bio Basic Inc). After 30 minutes on ice, the homogenates were centrifuged for 30 min (13,000 g, 4°C). The supernatants were collected for western blot analysis. Total protein concentrations were measuered using the bicinchoninic acid (BCA, Thermo Fisher) method. Proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes, which were subsequently blocked in 5% skim milk for 2 h at room temperature. The membranes were incubated with the following primary antibodies: rabbit anti-CaMK II (1:500, santa cruz), rabbit anti-NMDAR1 (1:1000,Epitomics), rabbit anti-GAP-43 (1:1000, Cell Signaling Technology), mouse anti-β-actin (1:4000; aBCAm) for overnight, then with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:4000, Proteintch) and anti-mouse secondary antibody (1:4000,Proteintch) for 2 h at room temperature.The immunopositive bands were detected by using an enhanced chemiluminescent substrate (Thermo Fisher) and a Bio-Rad ChemiDoc XRS digital documentation system (Bio-Rad). The band density was quantified using Image J software. The amount of protein expression is presented relative to the levels of β-actin.