Analysis of vascular perfusion, vessel density, tumor cell proliferation, apoptosis and hypoxia. (A) Tumors were harvested at completion of the study and examined by hematoxylin and eosin staining. Just before harvest, mice were infused with RCA-Lectin and hypoxia probe. Nuclei were counterstained with DAPI. RCA-Lectin localized the perfused vessels, CD31 localized microvascular endothelial cells, and the merged picture shows perfusion of total vessels in the field. Quantitation was performed with the use of Bioquant Image Analysis (Bioquant, Nashville, TN). Proliferating cells within the tumor were assessed by immunohistochemical detection of Ki-67 protein and quantified as described. All values are expressed as mean plus or minus SEM. *P < .01 by Student t test. Ki-67 pictures were taken with Carl Zeiss Invertoskop microscope with a 4×/0.12 NA objective and 10× eyepiece. Photomicrographs were taken using a Nikon Coolpix 5000 camera and a Nikon Eclipse E400 microscope with a 10× eyepiece. Magnification was as 40×/0.75 NA objectives. (B) sEphB4-HSA–treated tumor and adjacent normal tissue vessel density and perfusion. Dotted line demarcates the skin showing autofluorescence. Vessels in the subcutaneous tissue (subcutis) and the margin of the tumor show perfusion. Deeper tumor tissue shows progressive decrease in the vessel density and even greater decrease in vessel perfusion. (C) Gene expression analysis of tumor tissues was performed by quantitative PCR for VEGF, VEGFR1, VEGFR2, PDGF-β, and Dll4. Gene expression levels were corrected for β-actin levels.