Immunohistochemistry

Dcx/Ki67 staining. Slides were thawed for 10 min at room temperature then rehydrated with 1X PBS. Antigen retrieval was performed by incubating the slides in citrate buffer at 95°C for 15 min. Slides were cooled to room temperature before incubation with 5% NDS in 0.03% Triton X-100 for 1 hour at room temperature and then incubated with goat anti-Ki67 (1:500, Abcam, Cambridge, UK) and mouse anti-doublecortin (Dcx) (1:200, Santa Cruz Biotechnology, Texas, USA) antibodies in 5% NDS in 0.03% Triton X-100 at 4°C overnight. Slides were incubated in secondary antibodies (donkey anti-goat Alexa Fluor 488 and donkey anti-mouse Alexa Fluor 568; 1:400 in PBS, Invitrogen, Oregon, USA) for 1.5 hours at room temperature and then incubated with DAPI (1:10,000, Invitrogen, Oregon, USA) for 10 min. Slides were coverslipped using Vectamount mounting media (Vector, California). Sections were imaged every 200 μm beginning at the crossing of the corpus callosum and ending at the appearance of the anterior commissure. Approximately four to five sections were imaged per brain at a ×20 magnification.