Anti pax6

Mouse embryos were transcardially perfused with PBS, followed by 4% paraformaldehyde in PBS. Brains were removed and post-fixed overnight with 4% paraformaldehyde in PBS. Coronal sections (thickness of 80 μm) were cut with the vibratome VT1200 (Leica). Sections were permeabilized with 0.25% Triton X-100 in PBS and then blocked with PBST containing 2% bovine serum albumin (Sigma). Sections were incubated at 4 °C overnight with biotinylated HA-binding protein (b-HABP) (1:400, Hokudo), anti-NCAN (sheep, 1:400, R&D), anti-TNC (rat, 1:400, R&D), anti-Pax6 (rabbit, 1:400, Invitrogen), anti-Tbr2 (mouse, 1:400, Wako), anti-Nestin (mouse, 1:400, Millipore), anti-NeuN (rabbit, 1:1000, Proteintech), anti-Ctip2 (rat, 1:400, abcam), and Alexa Fluor 488-conjugated anti-GFP (rabbit, 1:1000, Invitrogen). After washing, sections were incubated with the appropriate Alexa Fluor conjugate secondary antibody and streptavidin (Thermo Fisher), followed by cell nuclear staining with DAPI (0.1 μg/mL). Images were captured with the AX R confocal microscope (Nikon) or LSM 710 NLO confocal microscope (Carl Zeiss). In a three-dimensional reconstruction, images were captured using the Z-stack function of the confocal microscope, and Z-projection and orthogonal views were obtained by ImageJ FIJI software.