Rabbit α activated caspase 3

Cells were fixed for 3 min in −20°C methanol or 8 min in 20°C 4% paraformaldehyde. Primary antibodies used in this study include rabbit α-NuMA and rat α-BrdU (Abcam, Cambridge, MA), mouse α-p150^glued and rat α-β4 integrin (BD Biosciences, San Jose, CA), mouse α-pankeratin (AE13), mouse α-Gata 3 and rat α-α tubulin (all from Santa Cruz Biotechnology, Dallas, TX), m α-β-tubulin (Sigma), rabbit α-keratin 6 (Covance), chicken α-keratin 5/14 (lab generated), rabbit α-phospho histone H3 and rabbit anti-pSMAD1/5 (Cell Signaling Technology, Danvers, MA), and rabbit α-activated caspase 3 (R&D Systems, Minneapolis, MN). Images were collected using a Zeiss motorized Axio Imager Z1 fluorescence microscope with Apotome attachment, an AxioCam MRm camera, a 10x, 20x, 40x, and 63×/1.4 numerical aperture (NA) Plan Apochromat objective, Zeiss Immersol 518F oil, and AxioVision Digital Image Processing Software. Photoshop (Adobe) and ImageJ software were used for postacquisition processing.

Embryos were fixed in 4% PFA in PBS buffer at 4 °C for 2.5–4 hours, cryoprotected, and sectioned at a thickness of 16 μm. The sections were incubated with primary antibodies overnight at 4 °C and then incubated with secondary antibodies for 2 hours at room temperature. The primary antibodies used were goat polyclonal α-NEUROG1 (1:700, Santa Cruz), rat monoclonal α-RFP (1:1000, ChromoTek), rabbit polyclonal α-SOX2 (1:500, abcam), mouse monoclonal α-TUJ1 (1:1000, Covance), goat polyclonal α-NEUROD1 (1:1000, Santa Cruz), rabbit α-activated caspase 3 (1:1000, R&D Systems), goat polyclonal α-SOX2 (1:700, Santa Cruz), rabbit polyclonal α-SOX2 (1:500, Millipore), and goat polyclonal SOX10 (1:100, Santa Cruz). The secondary antibodies used were Alexa Fluor 488 donkey α-goat (1:1000), Alexa Fluor 488 donkey α-rabbit (1:1000), Alexa Fluor 555 donkey α-rabbit (1:1000), Alexa Fluor 555 donkey α-rat (1:1000), Alexa Fluor 647 donkey α-mouse (1:1000), Alexa Fluor 647 donkey α-goat (1:1000).