Immunohistochemistry was carried out as previously described (24 (link)). The following primary antibodies were used at room temperature for 1 hour; CA9 (M75, 1/50); HIF1 (BD Biosciences, USA, 1/100); pimonidazole (Hypoxyprobe-1; Chemicon International, USA, 1/50); CD34 (MCA1825, AbD Serotec, USA, 1/50), cleaved caspase 3 (RnD systems, USA, 1/2000), CAXII (1/1000 (34 (link))) and KI67 (M7240, Dako, Denmark, 1/50). Slides were incubated with the anti-rabbit/anti-mouse secondary antibody (Dako, Denmark) for 30 minutes at room temperature and washed in PBS. DAB (Dako, Denmark) was applied to the sections for 7 minutes. The slides were counterstained by immersing in haematoxylin solution (Sigma-Aldrich) for 20 seconds and mounted with Aquamount (VWR, USA). Secondary only control staining was done routinely, these were negative. One section from each xenograft was analysed for each stain. Slides were scored by two researchers blinded to groupings (as a percentage or index based on intensity and percentage positive) or analysed quantitatively by image analysis in imageJ using colour deconvolution as described previously (35 (link)). Where scores differed sections were reviewed and a consensus result decided.