Anti brn2

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).

Immunohistochemistry was performed as described previously with slight modifications (Kawasaki et al., 2000 (link); Toda et al., 2013 (link)). Sections were permeabilized with 0.3% Triton X-100 in PBS and blocked with 2% bovine serum albumin (BSA), 0.3% Triton X-100 in PBS. For quadruple-staining and immunostaining with Ki-67, sections were subjected to microwave antigen retrieval in a sodium citrate solution. The sections were then incubated overnight with primary antibodies, which included anti-Hop (Santa Cruz Biotechnology, RRID:AB_2687966), anti-HopX (Atlas Antibodies, RRID:AB_10603770), anti-Tbr2 (R&D Systems, RRID:AB_10569705; Abcam, RRID:AB_778267), anti-Pax6 (Millipore, RRID:AB_1587367), anti-Ki-67 (Thermo Fisher Scientific, RRID:AB_10853185), anti-cleaved caspase 3 (BD Pharmingen, RRID:AB_397274), anti-Brn2 (Santa Cruz Biotechnology, RRID:AB_2167385), anti-Ctip2 (Abcam, RRID:AB_2064130), and anti-GFP antibodies (Nacalai Tesque, RRID:AB_2313652; Medical and Biological Laboratories, RRID:AB_591819). After incubation with secondary antibodies and Hoechst 33342, the sections were washed and mounted.