Chicken anti neun

For immunohistochemistry, virus injection, neuronal cultures and Western Blots, the following are used: goat anti-Nptx2 (Santa Cruz, sc-12125), chicken anti-NeuN (Millipore, abn91), rabbit anti-myc-tag (OriGene, TA100029), rabbit anti-parvalbumin (Abcam, ab11427), biotinylated WFA (Sigma-Aldrich, L1516), mouse anti-6X his-tag (Abcam, ab18184), Hoechst (Thermo Fisher, H3570), his-tag NPTX2 protein (R&D Systems, 10889), chABC (Sigma-Aldrich, C3667), test hyase (Sigma-Aldrich, H3506), strep hyase (Sigma-Aldrich, H1136), and lenti-cmv-Nptx2 lentivirus (10⁷ TU/mL, OriGene, MR206833L1V).

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).