Immunohistochemical analysis of tumor tissue sections. (A) Left panel: representative image of a whole tumor section stained with anti-CD31 antibody (blue), Hoechst 33342 (gray), and TUNEL (red) to label vasculature, nuclei and apoptosis, respectively. Vast red areas in tumor tissue represent continuous apoptosis regions, referred to as "necrosis". Middle panel: a magnified image of a viable area in tumor tissue section shown in the left panel; a blue arrow indicates a CD31 positive blood vessel; a red arrow indicates a TUNEL-positive apoptotic cell. Right panel: representative image of a viable area in the tumor tissue section stained with Hoechst 33342 (grey), anti-CD31 antibody to label blood vessels (a blue arrow) and anti-Ki67 antibody to label proliferating cells (a purple arrow). Scale bars: 150 μM. (B) Quantification of necrosis (a and d), apoptosis (b and e) and vascularization (c and f) in tumor tissue sections. Apoptosis is shown as % TUNEL-positive pixels within viable tumor tissue. The median distance from viable tumor tissue to the nearest vessel reflects microvessel density; the greater the vessel distance, the lower the microvessel density and vice versa. (C) The percentage of Ki67-positive pixels in viable tumor tissue. Bars in B and C represent the mean ± SE calculated from 4-5 tumors per treatment group and asterisks denote a statistically significant difference (p < 0.05) between the indicated groups. (D) Mean distribution of Ki67 positive pixels relative to CD31-stained vessels mapped in tumor tissue sections. The IHC analysis was completed twice using different cryosections to assure consistency of the results.