Immunohistochemical Analysis of HIF-1α and CAIX

Immunohistochemistry was performed on representative tissue block sections, chosen using hematoxylin and eosin stainings. Commercial monoclonal mouse antibody against HIF-1alpha (NB100-105 IgG2b, Clone H1alpha67, Novus Biologicals, Littleton, CO) has previously been validated for use in formalin-fixed paraffin-embedded material [24 (link)–26 (link)] and was used at a dilution of 1:300. Dako Envision flex kit (Dako, Copenhagen, Denmark) with high-temperature antigen retrieval in Tris-EDTA buffer for 20 min (pH 9.0) was used for detection of antibody reaction and diaminobenzidine (Dako basic DAB-kit) as a chromogen. Dako Autostainer (Dako) was used for the stainings.
The previously described monoclonal antibody M75 was used to recognize the N-terminal domain of human CAIX [27 (link)], with normal rabbit serum acting as negative control. Immunohistochemical staining was performed using automated Lab Vision Autostainer 480 (ImmunoVision Technologies Co., Brisbane, CA) and polymer-based Power Vision+™ Poly-HRP IHC Kit reagents (ImmunoVision Technologies, Co.) in room temperature, as described earlier [28 (link)].
Three series of controls (primary antibody omitted, primary antibody replaced with mouse primary antibody isotype control, and staining with irrelevant CD3-antibody) were performed. Specimens of ischemic intestine were used as positive controls for HIF-1alpha staining.