Immunolabeling for Neurogenesis Ablation

To confirm neurogenesis ablation, immunolabeling against doublecortin (DCX), a marker for immature neurons, was performed at the conclusion of all behavioral procedures as previously described (Wang et al., 2008 (link); Denny et al., 2012 (link)). Briefly, animals were given an overdose of ketamine/xylazine, then transcardially perfused with 0.1M phosphate buffered saline (PBS) followed by ice cold 4% paraformaldehyde (PFA) in PBS. Brains were dissected and postfixed overnight in 4% PFA followed by cryoprotection in 30% sucrose in PBS at 4°C. Serial 35-μm sections were then collected and stored in 0.02% NaN₃ in PBS at 4°C. Every sixth section was washed, incubated in H₂O₂ to quench endogenous peroxidases, blocked with normal donkey serum, and incubated overnight in primary antibody at 4°C (goat anti-DCX, 1:500, SCBT sc-8066; Dallas, TX). Following washes, sections were incubated in secondary antibody (biotinylated donkey anti-goat, 1:250, Jackson Immunoresearch; West Grove, PA) for 2 h at room temperature, treated with avidin-biotin-peroxidase complex, and exposed to 3,3′ diaminobenzidine substrate (Vector Labs; Burlingame, CA). Sections were then slide mounted, dehydrated through an ethanol series, and coverslipped with Permount before being imaged on an AxioObserver A.1 (Zeiss; Oberkochen, Germany).