Mixed Multiplex Staining: Automated RNAscope™ and OPAL™ for Multiple Targets

Abstract

The application of multiplex immunofluorescent detection of proteins, mRNAs, or both in combination has contributed to our understanding of biomarker expression and distribution within normal and pathological diseased tissues. In combination with whole-slide scanning these techniques permit the visualization of multiple biomarkers, their distribution within normal and pathological tissues, and their spatial and proximal distribution to each other in intact tissue sections. The expression of multiple biomarkers can now be used to accurately phenotype specific cell types, e.g., immune cells which rely on the expression of multiple biomarkers for precise identification. The availability of antibody reagents to detect specific protein targets, range of targets in specific technology platforms, and tissue fixation may mean that one technology may not be suitable for visualizing all biomarkers of interest. Here I demonstrate that a mixed multiplex approach for multiple protein and mRNA can be delivered on automated platforms suitable for high-throughput environments, and predict that mixed multiplex approaches will be important in future to compensate for tissue fixation differences, biomarker availability, and technology platform limitations.