*4.6. Western Blot Analysis*

Nrf2 and BDNF levels were assessed by Western blotting as described in the previous study [16]. Briefly, animals (*n* = 5 per group) were sacrificed 24 h after ischemia/reperfusion under deep anesthesia with a mixture of 75 mg/kg alfaxalone and 10 mg/kg xylazine on the 56th day of diet feeding. The left hippocampus was homogenized briefly, and the nuclear and cytosolic fractions were separated using extraction kits following the manufacturer's instructions (Abcam). Homogenized proteins were loaded onto sodium dodecyl sulfate polyacrylamide gel electrophoresis. Thereafter, the gel was transferred onto a nitrocellulose membrane (Pall Crop, East Hills, NY, USA), and the membrane was incubated with rabbit anti-Nrf2 (1:1000, Abcam) and rabbit anti-BDNF (1:5000, Abcam). Protein bands were visualized using a chemiluminescence solution (GE Healthcare, Buckinghamshire, UK) and were normalized versus laminin A + C and β-actin levels, respectively, as demonstrated in the previous study [20].
