Imaging Mass Cytometry for In Situ Immune Profiling.

The complexities and cellular heterogeneity associated with tissues necessitate the concurrent detection of markers beyond the limitations of conventional imaging approaches in order to spatially resolve the relationships between immune cell populations and their environments. This is a necessary complement to single-cell suspension-based methods to inform a better understanding of the events that may underlie pathological conditions. Imaging mass cytometry is a high-dimensional imaging modality that allows for the concurrent detection of up to 40 protein markers of interest across tissues at subcellular resolution. Here, we present an optimized staining protocol for imaging mass cytometry with modifications that integrate RNAscope. This unique addition enables combined protein and single-molecule RNA detection, thereby expanding the utility of imaging mass cytometry to researchers investigating low abundance or noncoding targets. In general, the procedure described is broadly applicable for comprehensive immune profiling of host-pathogen interactions, tumor microenvironments and inflammatory conditions, all within the tissue contexture.