Spatial immunophenotyping of FFPE tissues by imaging mass cytometry

The immune compartment of a tissue is dynamic, changing to respond to infections, tumors, or therapeutic interventions. Within tissues, local microenvironments provide interaction partners and cytokines that can gear immune cells into distinct functional states. Thus, it is not just the immune composition of a tissue, but also the relative localization of immune cells that determines the outcome of a response. Conventional techniques like immunohistochemistry (IHC) have been used to describe infiltration of immune cells and their relative position within tissues. However, these technologies are limited on the number of targets that can be simultaneously imaged. Here, we describe a simple protocol using imaging mass cytometry (IMC) for immunophenotyping formalin-fixed, paraffin-embedded (FFPE) tissues. IMC has a 1-μm resolution and allows simultaneous detection of up to 40 targets, overcoming limitations of traditional methods. In this protocol, we detail the staining procedure, offer an example of a murine FFPE antibody panel for immunophenotyping, and additionally provide suggestions for initial image analysis. The herein presented workflow facilitates the characterization of immune niches and can be used to assess their alterations throughout immune responses or therapeutic interventions. With minimal alterations, this approach can be used on clinically relevant samples or animal models to investigate specific immune responses and better understand disease progression or treatment dynamics.