Supporting Figures

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Fig. 6. Strategy for construction of ROSA26 ODD-Luc/+ mice. (A) Schema for homologous recombination. Shown are locations of Southern blot probes and PCR primers used for screening. (B) Representative PCR analysis of ROSA26 Luc/+, ODD-Luc/+, and +/+ mice by using primers that detect the presence of both ODD-Luc and luciferase (Top) or specific for ODD-Luc (Center). Gabra (Bottom) was used as an internal control.

Fig. 7. Bioluminescent imaging of ODD-Luc mice before and after renal artery ligation. Representative bioluminescent images (posterior view) of a ROSA26 ODD-Luc/+ mouse breathing room air before and immediately after left renal artery ligation. Color bar indicates photons·cm²·sec·steradian with minimum and maximum threshold values.

Fig. 8. Hypoxyprobe staining of mouse tissues. Representative sections of kidneys (×4 and ×10 magnification) (A) and indicated organs (×10 magnification) (B) obtained from mice breathing room air or exposed to 8% oxygen for 4 h. Hypoxic cells in histological sections were detected by using a Hypoxyprobe-1 Kit (Chemicon) according to the manufacturer’s instructions. Briefly, mice were injected with 60 mg/kg of pimonidazole hydrochloride (Hypoxyprobe-1) 1 h before being killed. Tissues were harvested and placed immediately into 10% buffered formalin acetate (Fisher Scientific). Protein adducts indicative of hypoxia were detected with Hypoxyprobe-1 monoclonal antibody, biotin-conjugated F(ab')2 fragment of a rabbit anti-mouse IgG, and peroxidase-conjugated streptavidin. Sections were counterstained with hematoxylin.

Fig. 9. Bioluminescent region of interest analysis. (A) Bioluminescent images of ROSA26 ODD-Luc/+ mice (Upper) and ROSA26 Luc/+ mice (Lower) breathing 21% or 8% oxygen. Color bar indicates photons·cm²·sec·steradian with minimum and maximum threshold values. Photon flux in particular regions of interest (circles) was counted and plotted in B. Filled bars, 21% oxygen. Hatched bars, 8% oxygen. Note that signal strength was not compromised in ROSA26 Luc/+ mice by hypoxia.