Immunohistochemistry of BrdU and NeuN

Immunohistochemistry was performed as described previously [6 (link), 18 (link)]. After anesthetization, all mice were perfused with 4% paraformaldehyde containing 0.5% picric acid. The brains were removed, fixed overnight, transferred to 30% sucrose, and stored at 4 °C. Coronal sections (14 μm) were generated using a cryostat. Consecutive sections were boiled in citrate buffer solution for 5 min and incubated with 2 N HCl at 37 °C for 30 min, followed by incubation in a blocking solution. The sections were incubated overnight with a monoclonal rat anti-BrdU primary antibody (1:5000; Novus Biologicals, Littleton, CO) and a monoclonal mouse anti-NeuN primary antibody (1:500; Millipore, Hayward, CA) in the blocking solution. Subsequently, the sections were incubated for 2 h with Alexa Fluor 594-conjugated goat anti-rat IgG (1:500; Invitrogen, Grand Island, NY) and Alexa Fluor 488-conjugated goat anti-mouse IgG (1:500; Invitrogen).