Fig. 2. Embryonic lens hypoxia visualized using the bioreductive marker pimonidazole. E17 lenses were incubated in pimonidazole in vitro (A–C) in solutions equilibrated with 21%, 2% or 0% O₂. Formation of pimonidazole–protein adducts was visualized immunocytochemically (see text for details). In 21% O₂ (A), no adducts were detected. However, adducts were detected in the cortex of lenses incubated in 2% O₂ (B), and intense cortical staining was present in lenses incubated in 0% O₂ (C). In vivo treatment with pimonidazole (D–F) resulted in strong cortical immunostaining throughout development. When pimonidazole was omitted from the intraocular injection solution, no immunostaining was observed (G), confirming the specificity of the pimonidazole antibody. Scale bar, 250 µm.