Anti phospho histone h3 ph3

The primary antibodies used were anti-RC2 (1:1, Iowa Hybridoma), anti-β-catenin (rabbit polyclonal, 1:1,000;), anti-GFP (chicken, 1:1,000; Abcam), anti-BrdU (mouse monoclonal, 1:50; BD Biosciences), anti-phospho-histone H3 (PH3) (rabbit polyclonal, 1:200; Millipore), anti-Tbr2 (1:500, AB23345, Abcam), anti-Ctip2 (1:500, AB18645, Abcam), anti-Cux-1 (1:100, SC13024, Santa Cruz Biotechnology), anti-Tbr1 (1:500, AB31940, Abcam) and anti-Brn1 (1:1,000, gift from A. Ryan, McGill University)10 (link). Secondary antibodies were AlexaFluor 488 or Cy3-conjugated (Invitrogen and Jackson ImmunoResearch). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (Sigma). Electroporated mouse brains were removed and fixed with 4% paraformaldehyde overnight at 4 °C. Brains were then embedded in 3% agarose and sectioned at 50 μm with a Vibratome (VT1000S; Leica Microsystems). Sections were blocked with PBS/10% goat serum/0.2% Triton X-100 for 1 h, and incubated in primary antibodies (see above) overnight at 4 °C. After three washes in 1 × PBS, sections were incubated with secondary antibodies (1:1,000) at room temperature for 2 h, washed and mounted with Citifluor anti-fading solution (Agar). For BrdU labelling, BrdU (50 mg kg⁻¹) was injected intraperitoneally into pregnant mice 1 h prior to brain tissue processing.