N phenylthiourea

Wild adult spotted gar were collected from the Atchafalaya River Basin, Louisiana. Spotted gar broodstock were injected with Ovaprim© (0.5 ml/kg) to induce spawning and cultured in a 2-m diameter tank containing artificial spawning substrate. Mean total length/weight for female and male broodstock was 691mm/1235g and 571mm/616g, respectively. Fertilized eggs from a group spawn were shipped to the University of Oregon and arrived two days after fertilization. Embryos were manually dechorionated with forceps and raised in fish water (1ppt salinity) at 24°C in a 14h light, 10h dark cycle. To inhibit melanin formation, 0.015 g/l N-Phenylthiourea (Sigma-Aldrich) was added to the fish water without any signs of developmental delay or malformations. Developmental stages were determined following (Long and Ballard, 2001 (link)). Animals were handled in accordance with good animal practice as approved by the University of Oregon Institutional Animal Care and Use Committee (Animal Welfare Assurance Number A-3009-01, IACUC protocol 12-02RA).

Prior to injection, HCT8 peak and PtcDN cells were treated for 48h with IgG isotype control or 5E1 antibodies. Then, 9×10⁵ cells were resuspended in serum-free medium and stained with lipophilic dyes DiO or DiD for 20 minutes at 37°C (ThermoFisher). Cells were then washed and resuspended in 30 μL of PBS. For zebrafish xenotransplantation, 48 hours post-fecundation (hpf) zebrafish embryos were anaesthetized with tricaine (Sigma-Aldrich) and 20 nL of cell suspension (approximately 300 labeled human cells) were injected into the perivitelline cavity of each embryo. The embryos were then placed at 30°C for 24 hours and allowed to recover in the presence of N-phenylthiourea (Sigma-Aldrich) to inhibit melanocyte formation. For imaging and metastasis assessment, zebrafish embryos were anaesthetized with tricaine and imaged using an Axio Observer Zeiss microscope (Zeiss).