Anti islet 1

Co-immunoprecipitation was conducted using a Co-IP assay kit (Merck Millipore, DA, Germany), and the total protein concentrations of the cell lysates were measured with a BCA assay. An anti-islet-1 (Abcam, Cambridge, MA, UK) antibody was used to pull down islet-1, and an anti-IgG antibody was used as a negative control. Protein collection was analysed by Western blot using an anti-Gcn5 (Epigentek, Farmingdale, NY, USA) antibody to detect its existence in islet-1-recruiting proteins.

P. vitticeps embryos at oviposition were fixed overnight in 4% paraformaldehyde (PFA) in PBS at 4°C, dehydrated though a series of methanol/PBS solutions (25, 50, 75, and 100% methanol), and stored at −20°C until hybridization or immunofluorescence. Whole mount in situ hybridization (WMISH) was performed according to our previously published protocol (Di-Poï et al., 2010 (link)) at a temperature of 68°C. New species-specific digoxigenin-labeled antisense riboprobes corresponding to P. vitticeps Dlx2 (831 bp, 3′ UTR region) and Sox10 (837 bp, 3′ UTR region) genes were designed based on publicly available P. vitticeps genome sequence (Georges et al., 2015 (link)). Corresponding sense riboprobes were used as negative controls. For immunofluorescence, embryos were embedded in paraffin following alcohol dehydration and then sectioned at 7 μm. Staining was performed as previously described (Di-Poï and Milinkovitch, 2013 (link)) using heat-induced epitope retrieval, primary antibodies known to recognize reptile and/or chicken epitopes (anti-β-tubulin: 1:400, Thermo Fischer Scientific; anti-ISLET-1: 1:700, Abcam), and Alexa Fluor-conjugated secondary antibodies (Alexa Fluor-488 or−568, Life Technologies). Samples were mounted with Fluoroshield mounting medium (Sigma) containing 4′,6′-diamidino-2-phenylindole (DAPI).

For immunofluorescence, cells and heart sections were blocked with normal goat serum for 40 min, and then incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies (1:500 dilution) to protect from the light at room temperature for 1 h. The following primary antibodies were used: anti-Sca-1 (1:100, Santa Cruz), anti-Islet-1 (1:300, Abcam), Nkx2.5 (1:100, Santa Cruz), and GATA4 (1:100, Santa Cruz). The following secondary antibodies were used: Alexa Fluor Chicken-anti-Rat 647, Alexa Fluor Goat-anti-Mouse 488, Alexa Fluor Chicken-anti-Rat 594, and Alexa Fluor Goat-anti-Rabbit 594 (Invitrogen). Microscopic examination was performed and was randomly selected from each chamber. All images were obtained using a LSM-780 confocal microscope and were analyzed using ImageJ software.