Anti tbr2

Immunohistochemistry was performed as described previously with slight modifications (Toda et al., 2013 (link); Kawasaki et al., 2000 (link)). Coronal sections were permeabilized with 0.3% Triton X-100/PBS and incubated overnight with primary antibodies, which included anti-Tbr2 (Abcam, UK, RRID: AB_778267), anti-Pax6 (Covance, Princeton, NJ, RRID: AB_291612), anti-Ki-67 (Leica, Germany, RRID: AB_442102), anti-phospho-histone H3 (Millipore, Billerica, MA, RRID: AB_310016), anti-phosphorylated vimentin (Medical and Biological Laboratories, Japan, RRID: AB_592963), anti-cleaved caspase 3 (BD Pharmingen, San Diego, CA, RRID: AB_397274), anti-Ctip2 (Abcam, UK, RRID: AB_2064130), anti-FOXP2 (Atlas antibodies, Sweden, RRID: AB_1078908), anti-GFAP (Sigma-Aldrich, St. Louis, MO, RRID: AB_477010) and anti-GFP antibodies (Nacalai tesque, Japan, RRID: AB_2313652; Medical and Biological Laboratories, Japan, RRID: AB_591819). After incubation with secondary antibodies and Hoechst 33342, the sections were washed and mounted.
For triple immunostaining, after double immunostaining was performed as described above, the sections were incubated with biotin-conjugated anti-phospho-histone H3 antibody (Millipore, Billerica, MA, RRID: AB_310794) and subsequently with fluorescent-dye conjugated streptavidin.

Cryosections were incubated with primary antibodies diluted in PBS/Triton X-100 overnight at 4°C, rinsed with PBS, and incubated with secondary antibodies for 1 hour. Sections were counterstained with DAPI (1 µg/ml, Sigma) and mounted in SlowFade Gold (Invitrogen) or Vectashield H-1000 (Vector Labs). Antigen retrieval was performed prior to overnight incubation. Antibodies used were anti-TBR1 (1:200, Abcam), anti-CTIP2 (1:500, Abcam), anti-BRN2 (1:50, Santa Cruz), anti-TBR2 (1:200, Abcam), anti-SATB2 (1:200, Abcam), and anti-γ-tubulin (1:200, Sigma). Secondary antibodies used were goat-anti-rabbit Alexa 594 and goat-anti-mouse Alexa 488 (1:800, Invitrogen).