Immunohistochemical Evaluation of Tumor Markers

MCT1, MCT4, CAIX and Ki67 protein expression for U251 shMCT1 and U251 shCTRL tumours collected from NSG mice were evaluated by immunohistochemistry (IHC). IHC for MCT1 was performed using UltraVision LP detection system HRP Polymer (Thermo Fisher Scientific, Waltham, MA, USA), and for MCT4, CAIX and Ki67 using UltraVision Large Volume Detection System Anti-Polyvalent, HRP (Thermo Fisher Scientific), as previously described [21 (link),23 (link)]. Briefly, deparaffinised and rehydrated slides were submitted to heat-induced antigen retrieval for 20 min at 98 °C with 10 mM citrate buffer (pH 6.0). After endogenous peroxidase inactivation, incubation with the primary antibody was performed overnight for MCT1, and during 2 h for MCT4, CAIX and Ki67, at room temperature. The immune reactions were visualized with 3,3′-diamonobenzidine (DAB + Substrate System; Dako, Denmark) as a chromogen. The slides were counterstained with haematoxylin and mounted with Entellan^® (Merck-Millipore, Darmstadt, Germany). For each immunoreaction, a positive control was included.