Hypoxic Responses in Zebrafish Embryos

Zebrafish embryos were obtained from adults of the corresponding transgenic lines; transgenic fishes were outcrossed with wild type individuals to ensure fluorescent signal homogeneity in heterozygous offspring. Fluorescent embryos at 24 h post fertilization (hpf) were incubated for 2–5 days at 28°C into a hypoxic chamber (ProOx P110 Oxygen Controller, BioSpherix, Parish, United States) with controlled and constant 5% oxygen concentration (about 2 mg/L, corresponding to a moderate hypoxia). As controls, fluorescent sibs were kept at 28°C outside the hypoxic chamber, under normoxic conditions.
A set of hypoxia treatments was performed following the incubation times applied by Kajimura and colleagues (Kajimura et al., 2005 (link)) to re-check the expression of zebrafish igfbp1a and igfbp1b genes in hypoxic (oxygen at 2 mg/L) and normoxic conditions: 24 h of hypoxia, starting from 1 hpf; 24 h of hypoxia, starting from 24 hpf; 24 h of hypoxia, starting from 48 hpf (Kajimura et al., 2005 (link); Kamei et al., 2008 (link)).