Molecular-assisted immunohistochemical optimization

Summary histochemistry (IHC) is an essential tool in diagnostic surgical pathology, allowing analysis of protein subcellular localization. The use of IHC by different laboratories has lead to inconsistencies in published literature for several antibodies, due to either interpretative (inter-observer variation) or technical reasons. These disparities have major implications in both clinical and research settings. In this study, we report our experience conducting an IHC optimization of antibodies against five proteins previously identified by proteomic analysis to be breast cancer biomarkers, namely 6PGL (PGLS), CAZ2 (CAPZA2), PA2G4 (EBP1) PSD2 and TKT. Large variations in the immunolocalizations and intensities were observed when manipulating the antigen retrieval method and primary antibody incubation concentration. However, the use of an independent molecular analysis method provided a clear indication in choosing the appropriate biologically and functionally relevant “staining pattern”. Without this latter step, each of these contradictory results would have been a priori “technically acceptable” and would have led to different biological and functional interpretations of these proteins and potentially different applications in a routine pathology setting. Thus, we conclude that full validation of immunohistochemical protocols for scientific and clinical use will require the incorporation of biological knowledge of the biomarker and the disease in question.