2.6 Western blot
The immunoblotting-based procedures in this study complied with the recommendations of the British Journal of Pharmacology (Alexander, Roberts et al., 2018). The rats were sacrificed by decapitation after isoflurane anaesthesia, and bilateral hippocampal tissue from each group was quickly extracted in ice-cold radio-immunoprecipitation assay lysis buffer (Life Technologies Co. USA; Cat# 89901). Proteins extracted from the hippocampal tissue were loaded on a 10% SDS-PAGE gel and blotted onto a hydrophobic polyvinylidene fluoride transfer membrane (0.45 µm pore size, Millipore, Billerica, MA, USA, Cat# IPVH00010). After being blocked with 5% (w/v) non-fat milk in Tris-buffered saline with 0.1% Tween-20 for 1 h, the membranes were probed with primary antibodies in blocking buffer at 4°C overnight and incubated with the horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, USA; 1:2000 Cat# 7074, polyclonal anti-rabbit IgG, RRID: AB_2099233, Cat# 7076, polyclonal anti-mouse IgG, RRID: AB_330924) for 1 h at room temperature. Immunoreactive antigens were detected using chemiluminescence agents (Bio-Rad Laboratories, Western ECL Blotting Substrates, RRID: SCR_008426) and visualised using the ChemiDoc™ XRS System with Image Lab Software (RRID: SCR_014210, Bio-Rad Laboratories, USA). Optical densities were analysed using ImageJ software (RRID: SCR_003070, NIH, Bethesda, Maryland, USA). The primary antibodies were diluted in 5% bovine serum albumin blocking buffer as follows: rabbit polyclonal IgG anti-p-ERK1/2 (Cell Signaling Technology, USA; 1:1000, Cat# 9101, RRID: AB_331646), rabbit polyclonal IgG anti-total ERK1/2 (Cell Signaling Technology, USA; 1:1000, Cat# 9102, RRID: AB_330744), rabbit monoclonal IgG anti-BDNF (Abcam, Cambridge, USA; 1:1000, Cat# ab108319, RRID: AB_10862052). Mouse monoclonal IgG anti-β-actin (Proteintech, USA; 1:8000, Cat# CL488-66009, RRID: AB_2883225) was used as the loading control, and the data were expressed as relative normalised expression and analysed by an independent researcher.