Rabbit anti tbr2

Brains were fixed overnight in 4% paraformaldehyde (PFA) at 4°C, followed by submersion in 30% sucrose until sinking, as previously described (Silver et al. 2010 (link)). Brain cryostat sections (20 µm) were prepared and stored at −80°C until use. Sections were permeabilized with 0.25% Triton X-100 for 10 min and blocked with MOM block reagent (Vector laboratories) for 1 h at room temperature (RT). Sections were incubated with primary antibodies for 2 h at RT or overnight at 4°C. Sections were then incubated in species appropriate secondary antibodies and Hoechst for 15 min at room temperature. The following primary antibodies were used: rabbit anti-CC3 (diluted 1:200; Cell Signaling); rabbit anti-TBR2 (1:1,000; Abcam); rabbit anti-PAX6 (1:1,000; Millipore); mouse anti-TUJ1 (1:400; Covance). The following secondary antibodies were used: Alex Fluor 488 and Alex Fluor 594 (1:400; Invitrogen). Hoechst (Thermo Fisher Scientific) was used for nucleus counterstain. High-magnification images were captured using a Zeiss Axio Observer Z.1 microscope coupled with an apotome. Cortical thickness was measured with Zen software. Cell quantification was performed with ImageJ/FIJI. Three sections from anatomically comparable regions per embryo and three biological replicates from control (wild-type) and mutant alleles (Casc3^RRU345/+, Casc3^{RRU345/RRU345}, and Casc3^RRU345/Null) were quantified.