Brain Tissue Preparation and Immunostaining

Animals were perfused transcardially with phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA). Brains were dissected, post-fixed in 4% PFA for 12–24 hours, and placed in 30% sucrose for 24–48 hours. They were then embedded in Optimum Cutting Temperature (OCT, Tissue Tek) and stored at −80°C until sectioning. 60 μm floating sections were collected into PBS. For Cre line characterization and layer analysis, sections were incubated in 0.3% PBST and 10% donkey serum for 1 hour and then stained with mouse anti-NeuN (Millipore MAB377, 1:1,000), rat anti–Ctip2 (Abcam ab18465, 1:200), or rabbit anti-Cux1 (Santa Cruz SC-13024, 1:500) for 1–4 nights at 4°C in 0.3% PBST and 5% donkey serum. All sections washed 3×10 min in PBS and additionally stained with NeuroTrace Blue (1:1,000) in 0.3% PBST for 2 hours, followed by DAPI (1:10,000 of 5 mg/mL, Sigma-Aldrich) in PBS for 10–15 min, and then washed once more with PBS prior to mounting onto Superfrost Plus slides and coverslipping with Fluorogel (Electron Microscopy Sciences). The sections were imaged at 5× using a Leica Ariol Slide Scanner microscope with an SL200 slide loader, and scanner images were processed with custom software^{55 (link)}.