Satb2

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.

At 1, 2, 4, 6, 8 and 19 weeks PST, animals were anesthetized with ketamine/xylazine (100 mg/kg and 10 mg/kg, respectively) and perfused transcardially with 0.9% saline followed by 4% paraformaldehyde. The brain samples were dissected and post-fixed with 4% paraformaldehyde for 18 hr and equilibrated in 30% sucrose at 4°C. Coronal sections of 40 µm thickness were prepared for samples from one-, two- and four-week PST brains. As the dendrites became too large to be contained within 40 µm sections, samples from 6-, 8- and 19 week PST brains were prepared at 80 µm using a sliding microtome. Tissue was mounted onto glass microscope sides with glycerol mounting medium with DAPI. The sections were immunostained with the antibodies GFP (1:400, Molecular probes) and TdTomato (1:400, LifeSpan Biosciences, Inc) to increase the signal of human and chimpanzee transplanted neurons. CTIP2 antibody (1:200, Abcam) and SATB2 (1:200, Abcam) were used to characterize cortical layer identity of the transplanted cells (Figure 4—figure supplement 1A–D).