Detailed Vascular Characterization of Xenograft Tumors

Xenograft tumour tissue was cryo-sectioned (20 µm) and processed as previously described.^{6 (link)} Endothelial cells were stained with a monoclonal Armenian-hamster anti-mouse CD31 antibody (1:200, Abcam) or a fluorescein isothiocyanate (FITC)-labelled anti-CD31 antibody (1:200, clone 390, Biolegend), and apoptotic cells were detected with a rabbit anti-cleaved caspase-3 (Asp175) antibody (1:200, Cell Signaling). Pericytes were detected either by a rabbit anti-NG2 Chondroitin Sulphate Proteoglycan antibody (1:100, Millipore) or a rat anti-PDGF-R antibody (1:100, CD140b, eBioscience). Vascular endothelial (VE) cadherin was stained with a rat anti-CD144 (VE-cadherin) antibody (BD Pharmingen). The following secondary antibodies derived from goat were used to label the respective primary antibodies with fluorescent dyes: Alexa Fluor 647 anti-hamster, Alexa Fluor 594 anti-rat, Alexa Fluor 594 anti-rabbit and Alexa Fluor 488 anti-hamster (1:500, Thermo Fisher Scientific). Vascular leakage was quantified by analysing intravenously injected FITC-dextran (molecular weight 2000 kDa, Sigma), pimonidazole adducts were detected with a FITC-labelled anti-pimonidazole antibody to visualise hypoxia; both procedures have been previously described in detail^{6 (link),19 (link)}; nuclei were stained with 4,6-diamidin-2-phenylindol (DAPI).