After the behavioral experiment, the mouse brain was quickly removed, and hippocampal tissues (six per group) were placed in ice-cold saline. The tissues were homogenized with RIPA buffer and protease inhibitors for 10 min and then centrifuged at 12,000 rpm for 30 min at 4 °C. The supernatants of the samples were assayed for protein content, diluted with 1:4 sample buffer and heated at 95 °C for 5 min. The proteins were loaded onto 10% sodium dodecyl sulfate (SDS) polyacrylamide gels and then transferred onto PVDF membranes. The membranes were incubated with blocking buffer (5% nonfat milk and 0.1% Tween-20 in Tris-buffered saline [TBST]) for 1 h at 27 °C. The membranes were incubated with the following primary antibodies overnight at 4 °C: anti-GluA1 and anti-GluA3 (Abcam 1:2000), anti-GluA2 (Millipore 1:2000), anti-NR1 (Sigma 1:1000), anti-NR2A and anti-NR2B (Abcam 1:2000), anti-ERK (Abcam 1:1000), anti-CREB (Abcam 1:1000), and anti-PSD95 (CST 1:1000). After three washes with TBST, the membranes were incubated with IRDye 700DW- or 800DW-conjugated anti-mouse or anti-rabbit IgG (1:10,000) for 1 h at room temperature, washed with PBS, and scanned to detect fluorescence with the LI-COR Odyssey detection system.
The investigators were blinded to the group assignment of the animals in all of the aforementioned experiments.
The investigators were blinded to the group assignment of the animals in all of the aforementioned experiments.