Detection of Keap1, Nrf2, HO-1, Bcl2, Bax and cleaved-caspase-3 protein expression levels in the hippocampus using western blotting

Protein lysis buffer (RIPA Lysis Buffer) was added to the hippocampus homogenates. Subsequently, the protein concentration was detected using bicinchoninic acid protein assay and 10% SDS-PAGE gel electrophoresis was performed on the samples (60 µg of protein per lane). Proteins were then transferred onto PVDF membranes and blocked with blocking buffer (TBST buffer containing 5% skim milk powder) for 1 h at room temperature before the primary antibodies (1:1,000) were added and incubated overnight at 4˚C. Secondary antibodies (1:5,000) were then added onto the membranes after washing with TBST and incubated at room temperature for 1 h. ECL chromogenic solution was used to develop the bands after the membranes were washed. ImageJ (version 1.51j8; National Institutes of Health) was used to perform the western blotting densitometric analysis.