Perfusion, dissection, and immunofluorescence staining were conducted according to standard protocols as previously described [17 (link)]. The following are the antibodies used: mouse anti-BrdU (1:50 dilution; BD Pharmingen, Franklin Lakes, NJ, USA), rabbit anti-Cux1 (1:100 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-Phospho-Histone H3 (1:250 dilution; Millipore, Billerica, MA, USA), rabbit anti-Pax 6 (1:500 dilution; Covance, Princeton, NJ, USA), rabbit anti-Tbr2 (1:500 dilution; Abcam, Cambridge, UK), mouse anti-Tuj1 (1:500 dilution; Covance, Princeton, NJ, USA), rabbit anti-Gli3 (1:100 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), and rabbit anti-Cleaved Caspase 3 (1:300 dilution; Cell Signaling, Madison, WI, USA).
For 5-bromo-2-deoxyuridine (BrdU, Sigma, St. Louis, MO, USA) labeling, pregnant dams were treated with 50 μg/g BrdU by intraperitoneal injection for 4 h prior to dissection at E16.5. DiI labeling was conducted by placing small crystals of the lipophilic tracer (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine; Invitrogen, Waltham, MA, USA) in the neocortex to target the upper layer (2/3) and then remained in 4% paraformaldehyde (PFA). After 6 weeks, brains were sectioned at 100 μm, counterstained with bisbenzimide, mounted, and imaged.
For 5-bromo-2-deoxyuridine (BrdU, Sigma, St. Louis, MO, USA) labeling, pregnant dams were treated with 50 μg/g BrdU by intraperitoneal injection for 4 h prior to dissection at E16.5. DiI labeling was conducted by placing small crystals of the lipophilic tracer (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine; Invitrogen, Waltham, MA, USA) in the neocortex to target the upper layer (2/3) and then remained in 4% paraformaldehyde (PFA). After 6 weeks, brains were sectioned at 100 μm, counterstained with bisbenzimide, mounted, and imaged.