Molecular profiling of Pogona vitticeps embryos

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).