Western Blotting

Rats were killed by decapitation 24 h or 1 month after treatment (immediately after the behavioral tests) and the hippocampus were removed and homogenized 1:10 (w/v) in HEPES 20 mM, pH 7.4 buffer, as previously described by Dutra et al., [45]. For Western blotting assays, the hippocampal tissue was homogenized in complete radioimmunoprecipitation lysis buffer (RIPA) containing 50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 2 mM EDTA, 1% Igepal CA-630, 1 mM Na 3 VO 4, 50 mM NaF, 1 mM PMSF, 20 μg/mL pepstatin A, 20 μg/mL leupeptin and 20 μg/mL aprotinin. The lysate was centrifuged twice at 13,000 g for 10 min at 4°C, and the supernatant was collected for protein concentration determination and preparation for electrophoresis. Equal amounts of protein extract (20 μg) were loaded per lane and electrophoretically separated using 10% denaturing polyacrylamide gel electrophoresis (SDS-PAGE). Afterward, the proteins were transferred to nitrocellulose membranes using a Mini Trans-Blot Cell System (Bio-Rad Laboratories Inc., Hercules, CA, USA) following the manufacturer’s protocol. The membrane was blocked with 5% BSA in 0.05% TBST for 1 h at room temperature and then immunoblotted with the following antibodies: anti-β-actin (#3700, 1:1000, Cell Signaling Technology, Danvers, USA), anti-NMDAR1 (#4204, 1:1000, Cell Signaling Technology, Danvers, USA), anti-NMDAR2B (#MAB5220, 1:1000, EMD Millipore Corporation, Billerica, USA) and anti-GR (#SAB4501310, 1:500, Sigma-Aldrich) in blocking buffer at 4°C overnight. Following washing, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (1:25,000, Cell Signaling Technology, Danvers, MA, USA). The immunocomplexes were visualized using SuperSignal West Femto Chemiluminescent Substrate Detection System (Thermo Fischer Scientific, Rockford, IL, USA) and densitometric values were normalized using β-actin densitometric values. Protein levels were quantified by optical density using Image-J Software® and expressed as the ratio to β-actin represented by arbitrary units.