Immunohistochemistry
SECONDARY, POSITIVE
Indirect chromogenic immunohistochemical staining was performed on 5-μm thick paraffin-embedded tissue sections on coated slide glasses. We used primary antibodies against GLUT1 (RB-9052-P, Thermo Fisher Scientific, Waltham, MA, USA; dilution 1:500), GLUT3 (RB-9096-P, Immunologic, WellMed BV, Duiven, the Netherlands; 1:300), GLUT4 (ab654, Abcam, Cambridge, UK: 1:1000), HK1 (MA5-15,680, Invitrogen™, Thermo Fisher Scientific; 1:7500), HK2 (MA5-15,679, Invitrogen™, Thermo Fisher Scientific; 1:200), HIF1α (ab2185, Abcam; 1:250), MCT4 (anti SLC16A4, HPA046986, Sigma-Aldrich®, Saint Louis, MO, USA; 1:200), CA-IX (NB100-417, Novus Biologicals™, Bio-techne, Centennial, CO, USA; 1:250), VEGF (555,036, Pharmingen™, Becton Dickinson Biosciences, San Diego, CA, USA; 1:100), NIS (ABC1453, Merck Millipore, Burlington, MA, USA; 1:2000), and Ki-67 (M724001, DAKO Agilent, Santa Clara CA, USA; 1:25).Tissue sections were deparaffinized and rehydrated. Next, GLUT1, GLUT3, HIF1α, CA-IX, and Ki-67 staining was performed using a semi-automated immunostainer (Thermo Scientific, Labvision™ 488) following a standardized protocol according to manufacturer instructions. GLUT4, HK1, HK2, MCT4, VEGF, and NIS staining was performed manually. Detailed immunohistochemistry procedures are provided in the Supplementary data. In summary, antigen retrieval was performed, non-specific immunoreactivity was blocked, and slides were subsequently incubated with primary and secondary antibodies. Slides were incubated using 3,3-diaminobenzidine (DAB) as chromogen. Negative control samples were processed without primary antibodies. Positive control tissues were used as recommended by the manufacturer. Finally, slides were counterstained with hematoxylin and dehydrated.All slides were reviewed to quantify the expression of the immunohistochemical markers by evaluating the DAB staining by two members of the research team (EK and AE), who were blinded to the clinical data and [