2.4. Immunohistochemistry

To visualize the proliferating cells and differentiated neuroblasts in the hippocampus, immunohistochemical staining was conducted for Ki67 and doublecortin (DCX), respectively, as previously described [9]. Briefly, animals (n = 5 per group) were anesthetized with a mixture of 75 mg/kg alfaxalone and 10 mg/kg xylazine at 8 weeks after diet feeding and were perfused transcardially [10]. Frozen brain sections (30-μm thickness) were collected into six-well plates based on the mouse atlas by Franklin and Paxinos [28] between 1.46 mm and 2.46 mm posterior to the bregma.

Five sections located 90 μm apart were used for immunohistochemical staining with each antibody [10]; sections were incubated with rabbit anti-Ki67 antibody (1:1000, Abcam, Cambridge, UK), and a rabbit anti-DCX antibody (1:5000, Abcam) and the immunoreaction was visualized with nickel intensified 3,3′-diaminobenzidine tetrachloride (Sigma, St. Louis, MO, USA) in 0.1 M Tris-HCl buffer (pH 7.2). Sections were dehydrated and mounted on gelatin-coated slides in Canada balsam (Kanto Chemical, Tokyo, Japan).