Figure 3

Localization of HYPOX-4 and pimonidazole in transverse retinal sections from OIR mice.

OIR pups (P12) were treated with HYPOX-4 or pimonidazole and the spatial distribution of hypoxia was determined in retinal cross-sections. (A) HYPOX-4 dependent fluorescence activity indicated alternating regions of hypoxia in the inner retina overlapping with retinal avascularity (green); hypoxia was visualized in the inner plexiform and inner nuclear layers. Presumably, oxygen diffusion out of the major vessel indicated by the white arrow, inhibits increased azo/nitroreductase activities and consequently the retention of HYPOX-4. (B) Pimonidazole-adduct immunostaining confirmed retinal hypoxia in the inner plexiform and inner nuclear layers; additionally, this method detected hypoxia in the ganglion cell layer (red). (A,B) retinal nuclei were stained with DAPI (blue). Abbreviations: GCL = ganglion cell layer, IPL = inner plexiform layer, INL = inner nuclear layer, OPL = outer plexiform layer, ONL = outer nuclear layer.