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.