Zebrafish Embryonic Pharmacological Interventions

Camptothecin (Sigma # C9911) treatment was performed according to a previously described method [45] (link) with modifications. The compound (60 µM in 0.1% dimethyl sulfoxide [DMSO]) was applied to 48 hpf embryos, which were harvested at 57 hpf for the 3β-Hsd activity assay. The treatment of embryos with 2,3-butanedione 2-monoxime (2,3-BDM; Sigma #B0753) was as described in an earlier report [46] (link), except that dechorionated embryos were immersed in various concentrations of 2,3-BDM starting from 1.5 dpf. Norepinephrine treatment was performed by treating dechorionated embryos with 0.01, 0.1 or 1 mM Norepinephrine (Sigma A7257) freshly prepared in egg water. L-NAME treatment was performed by treating dechorionated embryos with freshly prepared 100 µM Nω-nitro-l-arginine methyl ester (l-NAME) (#N5751, Sigma) in egg water with 0.1% DMSO at 36 hpf. For l-arginyl-l-glycyl-l-aspartic acid (RGD; #G1269, Sigma) treatment, the peptide was reconstituted to 1 mM in filter-sterilized egg water and applied to dechorionated embryos at a final concentration of 100 µM at 26 hpf; embryos were collected at 2.5 dpf and fixed for histological assays.