Adult zebrafish were bred and maintained as described previously35 (link),41 (link). Embryos collected from the tanks were kept in water to reach the desired developmental stage for drug treatment. Embryo stage is given in hours post fertilisation (hpf). Wildtype (WT) zebrafish embryos at 24 hpf, 48 hpf and 72 hpf were exposed to mixtures of NA/EE (in ratios equivalent to Primodos) under different concentrations or DMSO only (0.2%). Drug testing and analysis were carried out as described previously30 (link),32 (link),34 (link),35 (link),41 (link). Briefly, embryos were hand dechorionated and exposed to the drugs or DMSO. For phenotypical analyses, embryos were fixed in 4% Paraformadehyde (Sigma-Aldrich) in 1x PBS at 96 hpf and for gene expression analyses, cell death and immunohistochemistry, embryos were fixed in 4% paraformaldehyde at 6 hr and 24 hr following treatment.
fli1:EGFP zebrafish embryos (obtained from the Zebrafish International Resource Center) were used to analyse the effects of the NA/EE mixtures on blood vessel growth using previously published protocols30 (link),32 (link),34 (link),35 (link),40 (link),41 (link). All animal research was licensed, approved and carried out following guidelines issued by the UK Home Office and University of Aberdeen Ethics Review Committee.
fli1:EGFP zebrafish embryos (obtained from the Zebrafish International Resource Center) were used to analyse the effects of the NA/EE mixtures on blood vessel growth using previously published protocols30 (link),32 (link),34 (link),35 (link),40 (link),41 (link). All animal research was licensed, approved and carried out following guidelines issued by the UK Home Office and University of Aberdeen Ethics Review Committee.
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