Mouse antibody to satb2

Embryos and pups were fixed for 5 to 8 hr in phosphate-buffered saline (PBS) containing 4% PFA (wt/vol), incubated overnight at 4°C with 20% sucrose in PBS (wt/vol), embedded in OCT compound (Sakura Finetek, Tokyo, Japan), and sectioned with a cryostat to obtain 14-µm-thick coronal sections. For detection of Rorb and BrdU, antigen retrieval was performed by autoclave treatment of the sections for 5 min at 105°C in 0.01 M sodium citrate buffer (pH 6.0). As primary antibodies, we used mouse antibody to BrdU (BD Biosciences, San Diego, CA), rabbit antibody to GFP (MBL, Nagoya, Japan), chicken antibody to GFP (Abcam, Cambridge, UK), rabbit antibody to Rorb (Diagenode, Leige, Belgium), mouse antibody to Rorb (Perseus Proteomics, Tokyo, Japan), goat antibody to Lhx2 (Santa Cruz, Santa Cruz, CA), mouse antibody to Satb2 (Abcam), goat antibody to KCNH5 (Santa Cruz), goat antibody to NetrinG1 (R&D systems, Minneapolis, MN) and rabbit antibody to Tbr1 (kind gift from R. Hevner, University of Washington). Immune complexes were detected with FITC–, TRITC– or Cy5–conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). For nuclear staining, we used 1 µg/ml propidium iodide (PI; Molecular Probes, Eugene, OR) and 2 µg/ml DAPI (Molecular Probes). Images were acquired using confocal microscopes (FV300 and FV1000; Olympus, Tokyo, Japan).

Primary antibodies and dilutions used were: rat antibody to CTIP2 (1:500, Abcam); mouse antibody to SATB2 (1:200, Abcam); rabbit antibody to SIRT1 (1:250, Millipore); rabbit antibody to CTIP1 (1:500, Abcam); rabbit antibody to GFP (1:500, Invitrogen); rabbit antibody to Ki67 (1:500, Abcam); chicken antibody to NESTIN (1:500, Novus Biologicals); mouse antibody to TuJ1 (1:500, Covance); and mouse antibody to MAP2 (1:500, Sigma). Alexa fluorophore conjugated secondary antibodies from Invitrogen were used at a dilution of 1:1000. Hoechst 33342 counterstain was used to visualize nuclei (1:3,000, Invitrogen).