Figure 2.

RENCA tumor cells do not express iNOS protein, although infiltrating macrophages do. Tumors were generated by subcutaneously injecting 2 × 10⁶ RENCA cells into the flanks of BALB/c mice (n = 6). Tumors were harvested 14 days (small without necrosis) or 28 days (with a necrotic core) after injection, paraffin-embedded, sectioned, and stained (A) with H&E and for HIF-1, the pan-macrophage marker F4/80, and iNOS. Scale bar = 100 μm. B: The mean vessel distance was estimated by CD31 staining. C: The distribution of macrophages per area was measured. D: The intensity of iNOS expression was evaluated by assigning an immunoreactive score. E: The generation of nitrites was measured. F: Expression of iNOS mRNA (green) was evaluated by in situ hybridization followed by staining for F4/80 (red). NC, scrambled probe as negative control. Scale bar = 20 μm. **P < 0.01, ***P < 0.001 relative to the small, non-necrotic tumors.