Immunofluorescence

Mouse brains were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature (4 h to overnight), incubated in 20% sucrose at room temperature (4 h to overnight), and then embedded in OCT compound and frozen in liquid nitrogen. Frozen brain samples were sectioned at a thickness of 14 μm (Leica cryostat CM3050) and blocked with blocking buffer (5% donkey serum in PBS), and then incubated with primary antibodies diluted in blocking buffer at room temperature (1 h to overnight). The primary antibodies were visualized using Cy3-conjugated anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) or Alexa488-conjugated anti-rat or anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA. The primary antibodies used were against phospho-histone H3 (pH3) (Cell Signaling, Danvers, MA), γ-H2AX (Millipore, Billerica, MA), Sox2 (Santa Cruz Biotechnology, Santa Cruz, CA), βIII-tubulin (clone Tuj1; Covance, Princeton, NJ), ZO-1 (Invitrogen), cleaved-caspase-3 (Cell Signaling), γ-tubulin (Santa Cruz), Tbr2 (Abcam, Cambridge, UK), Brn1 (Santa Cruz), and Foxp2 (Abcam). Samples were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and analyzed using a confocal microscope (Zeiss LSM510) or a conventional microscope.