Anti psd 95

The tissues were homogenized in radioimmunoprecipitation (RIPA) buffer containing a protease inhibitor and phosphatase inhibitor. The protein concentration was determined using Bradford reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of the total protein (10–30 μg) were electrophoresed on 8–12.5% sodium dodecyl sulfate (SDS)–polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were incubated in 3% bovine serum albumin (BSA) solution for nonspecific blocking, followed by overnight incubation at 4 °C with the primary antibody. The following primary antibodies and dilutions were used: Anti-6E10 (cat no. SIG-39320; 1:1000 dilution; Biolegend), anti-Iba-1 (cat no. 106-20001; 1:1000 dilution; Wako), anti-synaptophysin (cat no. MAB-5258; 1:1000 dilution; Merck), anti-PSD-95 (cat no. GTX133091; 1:1000 dilution; GeneTex, Irvine, CA, USA), and anti-β-actin (cat no. A1978; 1:2000 dilution; Sigma-Aldrich) antibodies. Subsequently, the membranes were washed in PBS containing 0.1% Tween 20 and incubated with a 1:3000 dilution of horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature. The membranes were then washed and developed using an enhanced chemiluminescence (ECL) kit (Perkin Elmer, Waltham, MA, USA).

After electrophysiological experiment, the rats were sacrificed immediately and the following protocols reported previously with minor modification were conducted (Wang et al., 2017a (link); Yang et al., 2017 (link)). The hippocampus was removed at 0°C and homogenized in RIPA buffer which contained 1% PMSF (Solarbio, China). Lysates were then centrifuged at 12,000 rpm at 4°C for 20 min, and the supernatants were collected. Protein concentration was determined using the BCA Protein assay kit (Solarbio, China). After that, equal amounts of protein (40 ug/lane) for each sample were loaded and run on an 8–15% SDS-PAGE gel, which were transferred to 0.44 um polyvinylidene difluoride (PVDF) membrane (Millipore Corporation) at 4°C (BIO-RAD, United States). The PVDF membrane was blocked in Tris-buffered saline with Tween-20 (TBST) containing 5% skimmed milk for 1 h at room temperature. Next, the membranes were incubated with primary antibody overnight at 4°C (anti-SYP 1:10000, anti-PSD95, anti-NMDAR2B 1:2000, Genetex). After washing thrice with TBST, the PVDF membranes were subsequently incubated with secondary antibody (anti-mouse IgG HRP conjugate, anti-rabbit IgG HRP conjugate, 1:2000, Genetex) for 1 h at room temperature. Finally, a computerized chemiluminescent imaging system (Tanon Science & Technology, China) was employed to identify the protein band intensities.