Methods for tissue microarray construction were previously described (Eustace et al, 2013a (link)). Immunohistochemistry was carried out for CAIX and GLUT1 as per a previous protocol (Hoskin et al, 2003 (link)). HIF-1α staining was carried out using the Bond-Max Automated staining system (Leica Biosystems, Newcastle, UK). Samples were de-waxed and rehydrated, followed by antigen retrieval at pH 9.0 for 40 min at 100 °C. Endogenous peroxidase was blocked using 3% hydrogen peroxide solution. Primary antibody (mouse monoclonal HIF-1α, BD Biosciences 610959, Oxford, UK) was diluted to a 1 : 20 solution with diluent and incubated with samples for 15 min at room temperature. A negative control of IgG1 (Dako X0931, Cambridge, UK) was also used. Post-primary rabbit anti-mouse link reagent was applied (Bond Polymer Refine Detection System, Leica DS9800, Newcastle, UK), and samples were incubated for 8 min at room temperature. The anti-rabbit polymer-HRP detection reagent (Bond Polymer Refine Detection System, Leica) was then added and samples were incubated at room temperature for a further 8 min. 3,3′-diaminobenzidine tetrahydrochloride was added, and after a further 10 min of incubation samples were counterstained with haematoxylin.