Anti camkiiα

Homogenates of hippocampal tissue were prepared in RIPA buffer containing 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% sodium deoxycholate, 1% Triton X-00, 1 mM PMSF, 50 mM sodium fluoride, 1 mM sodium vanadate, 1 mM DTT, and protease inhibitors cocktails. PSD fractions were directly dissolved in 1× SDS-PAGE sample buffer. All the protein samples were boiled in 100 °C water bath for 10 min before western blot. The homogenates were resolved on SDS-PAGE and transferred to nitrocellulose membranes, which were incubated in the TBS buffer containing 0.1% Tween-20 and 5% milk for 1 h at room temperature before incubation with a primary antibody overnight at 4 °C. After wash, the membranes were incubated with an HRP-conjugated secondary antibody in the same TBS buffer for 1 h at room temperature. Immunoreactive bands were visualized by ChemiDocTM XRS + Imaging System (BIO-RAD) using enhanced chemiluminescence (Pierce) and analyzed with Image J (NIH). The primary antibodies used were as follows: anti-Histone H3, Cell Signaling (9715); anti-Calmodulin, Millipore (05-173); anti-CaMKIIα, Cell Signaling (11945); anti-p-CaMKIIα Thr²⁸⁶, Sigma (SAB4300228); anti-Acetyllysine, Cell Signaling (9441); anti-p-GluR1 Ser⁸³¹, Abcam (ab109464); anti-GluR1, Abcam (ab109450); anti-GST, Abmart (12G8); and anti-His, Abmart (10E2).

Immunofluorescent staining was performed by washing the brain sections with PBS 3 times (10 minutes each time) and incubating them with blocking buffer containing 7% normal donkey serum, 0.3% Triton X-100, and 0.05% sodium azide at room temperature for 1 hour. The sections were subsequently incubated with primary antibodies diluted in blocking buffer overnight at 4°C. After incubation, the brain sections were rinsed 3 times (10 minutes each time) with PBS and incubated with a fluorescent dye–conjugated secondary antibody for 1 hour at room temperature. After 3 washes (10 minutes each time) with PBS, the brain sections were incubated with DAPI. The primary antibodies used were the following: anti-c-fos (1:200, Rabbit, Cell Signaling Technology, Danvers, MA), anti-c-fos (1:500, Mouse, Santa Cruz Biotechnology), anti-CaMKIIα (1:200, Mouse, Cell Signaling Technology, Danvers, MA), anti-GABA (1:500, Rabbit, Sigma-Aldrich), and anti-NeuN (1:100, Rabbit, Cell Signaling Technology, Danvers, MA). The secondary antibody included antirabbit Alexa Fluor 488 (1:500, Invitrogen) or antimouse Alexa Fluor 555 (1:500, Invitrogen).