Mouse brains were fixed in 4% PFA in phosphate-buffered saline (PBS) overnight, incubated in 25–30% sucrose in PBS, embedded in OCT (SAKURA, USA), and stored at –80 °C until use. Brains were sectioned (14–16 μm) using a cryostat. For antigen recovery, sections were incubated in heated (95–100 °C) antigen recovery solution (1 mM EDTA, 5 mM Tris, pH 8.0) for 15–20 min, and cooled down for 20–30 min. Before applying antibodies, sections were blocked in 10% normal goat serum in PBS with 0.1% Tween-20 (PBT) for 1 h. Sections were incubated with primary antibodies at 4 °C overnight and visualized using goat anti-rabbit IgG–Alexa-Fluor-488 (Jackson ImmunoResearch, UK) and/or goat anti-mouse IgG–Alexa-Fluor-546 (Jackson ImmunoResearch, UK) (1:300, Molecular Probes) for 1.5 h at room temperature. Images were captured using a Leica digital camera under a fluorescent microscope (Leica DMI6000B).
Primary antibodies against the following antigens were used: BrdU (1:100) (Abcam, UK), Ki67 (1:300) (Abcam, UK), Sox2 (1:100) (Santa Cruz Biotechnology, USA), Pax6 (PRB-278P, 1:200, BioLegend), Tbr1 (1:2,000) (Abcam, UK), Tbr2 (1:500) (Abcam, UK), NeuN (1:3,000) (Abcam, UK), Satb2 (1:500) (Abcam, UK), Caspase3 (1:100, Abcam), and DAPI (D9542, 1:1000, Sigma), also see Supplementary Table S14 .
Primary antibodies against the following antigens were used: BrdU (1:100) (Abcam, UK), Ki67 (1:300) (Abcam, UK), Sox2 (1:100) (Santa Cruz Biotechnology, USA), Pax6 (PRB-278P, 1:200, BioLegend), Tbr1 (1:2,000) (Abcam, UK), Tbr2 (1:500) (Abcam, UK), NeuN (1:3,000) (Abcam, UK), Satb2 (1:500) (Abcam, UK), Caspase3 (1:100, Abcam), and DAPI (D9542, 1:1000, Sigma), also see Supplementary Table S