Figure 3. Morphology-based histological techniques to functionally assess the vasculature of tumours. (A) Fluorescent-micrographic image of the dense vascular network of an experimental tumour using CD31 immunohistochemistry. Pan-endothelial cell markers such as vWF, CD31, or CD34 can be used to determine the overall microvessel density (MVD), which is a global angioarchitectural readout of the average intercapillary distance. (B) Double labelling of blood vessels (CD31, brown staining) and proliferating cells (Ki67, dark nuclei) in a human glioblastoma. Co-localization of CD31 and Ki67 staining allows the detection of the few proliferating endothelial cells (arrows) among the many proliferating tumour cells. (C) Fluorescent microscopic triple staining of tumour cell nuclei (Hoechst stain, blue), blood vessels (CD31, red), and proliferating endothelial cells (Ki67, green) in an experimental human tumour growing in an immunocompromised mouse. The anti-murine Ki67 stains only proliferating mouse cells and not the xenografted human tumour cells allowing the specific detection of the proliferatin, angiogenic mouse blood vessel compartment. (D) Three-dimensional co-localization of an endothelial cell marker (CD31, green) and a mural cell marker (desmin, red) demonstrating the intense mural coverage in the quiescent subcutaneous vasculature of the mouse. Correspondingly, the degree of maturation of the tumour vasculature can be traced by employing appropriate mural cell markers (-smooth muscle actin, desmin, NG2, RGS5).