Amplification kit

Immunohistochemistry was performed on cut sections from formalin fixed paraffin embedded (FFPE) tumour tissue. 4 μm tissue sections were cut and de-waxed through histolene, and graded alcohols then into water. Antigen retrieval was performed using Dako high pH Target Retrieval Solution (Dako) for 3 min at 125 °C for Glut-1, HIF-1 α and CA-9. Slides were then loaded onto a Dako autostainer (Dako) for the following incubations: primary antibody for Glut-1 (1:200, Dako), HIF-1α (1:50, Novus Biologicals) or CA-9 (1:4000, Novus Biologicals) for 60 min at room temperature; antibody detection with Envison+ (Dako) rabbit (Glut-1 and CA-9) or mouse (Hif-1α) antibody for 60 min at room temperature; colour reaction with 3,3′-Diaminobenzidine (DAB) for 10 min at room temperature. Staining for Ki-67 was performed on a Ventana BenchMark Ultra (Roche Diagnostics, USA). Antigen retrieval was performed in a high pH Ultra cell conditioning solution (CC1, Roche Diagnostics) for 52 min followed by incubation with the Ki-67 antibody (SP6, Cell Marque, diluted at 1/50), at 36 °C for 32 min. Amplification kit (amplifiers A and B, Roche Diagnostics) and UltraView Universal DAB detection kit (Roche Diagnostics) were used in accordance with the manufacturer’s instructions for on-board detection. Slides were then removed from the autostainer, counterstained with hematoxylin, mounted and coverslipped.