Angiogenesis in WT and IFN-γ knockout tumors. (A) CD31 (PECAM-1) staining for qualitative analysis of microvessel density Paraffin-embedded tissue sections were subjected to deparaffinization, rehydration, and antigen unmasking as described in “Materials and methods.” At least 4 tumor specimens were stained for each genotype as follows: each slide was incubated with 5 μg/mL CD31 antibody diluted in 1% normal goat serum for 1 hour at room temperature, and the bound antibody was stained by means of the ABC immunohistochemistry kit with the use of streptavidin-HRP. The slides were counterstained in Harris hematoxylin and mounted. (B) Quantitation of angiogenesis in WT and IFN-γ knockout tumors. The tissues were observed under a bright-field microscope for quantitative evaluation of positively stained blood vessels or angiogenic areas at 200 × magnification. The numbers of fields quantitated are denoted as n values. The statistical significance for analysis was established with a paired t test as follows: Tax⁺IFN-γ^+/+ versus Tax⁺IFN-γ^+/- (P ≤ .11); Tax⁺IFN-γ^+/- versus Tax⁺IFN-γ^-/- (P ≤ .03); and Tax⁺IFN-γ^+/+ versus Tax⁺IFN-γ^-/- (P ≤ .002). Error bars correspond to SEM.