Immunohistochemical Analysis of Neurogenesis

For immunohistochemistry, tissue was prepared as previously described [41 (link),42 (link)]. In brief, mice were deeply anaesthetized with CO₂. Mice were transcardially perfused with PBS (pH 7.4) followed by 4% PFA (pH 7; Roti^®-Histofix, Karlsruhe, Germany). Brains were post-fixed in 4% PFA (pH 7; Roti^®-Histofix) for 12–72 h at 4 °C, then dehydrated in 30% sucrose in ddH₂O at 4 °C for 24–48 h. Brains were cut into 40 µm coronal sections using a Leica cryotome (Product CM1850, Leica Microsystems, Wetzlar, Germany). Immunostaining was used as previously reported [41 (link)]. In brief, free-floating coronal brain sections were washed 3 times in PBS (pH 7.4), then blocked in blocking solution (1% (w/v) BSA, 0.5% (v/v) Triton X-100 in PBS) and incubated with primary antibodies overnight at 4 °C. Antibodies were diluted in blocking solution (rabbit anti-Doublecortin. DCX, 1:1000; Abcam); chicken anti-NeuN (1:500; Millipore, Darmstadt, Germany). Upon incubation, sections were washed in PBS and incubated with secondary antibodies (donkey anti-rabbit IgG Alexa Fluor 555, AF555, labeled and goat anti-chicken IgY Alexa Flour 647, AF647, labeled (both Life Technologies, Carlsbad, CA, USA) diluted 1:500 in blocking solution for 2 h. Upon washing, sections were mounted on Superfrost glass slides (Carl Roth, Karlsruhe, Germany) with Fluoromount (Sigma, Darmstadt, Germany).