The tumor microenvironment (TME) is composed of tumor cells and surrounding stroma, including immune, mesenchymal, and vascular cells, as well as soluble factors. Traditional techniques to interrogate cellular interactions and infiltration are limited by the loss of tissue architecture or by labeling only a small number of antigens. Multiplex fluorescent immunohistochemistry (mfIHC) is a tissue staining technique that involves tyramide-based signal amplification to covalently bind a fluorophore to an antigen of interest, followed by removal of the primary and secondary antibodies, thereby permitting co-staining and localization of multiple antigens on a single sample. mfIHC retains spatial information, allowing for in-depth analysis of cellular phenotypes and their interactions within the TME. Described here is an overview of the mfIHC experimental design, staining, and image analysis in murine tissues utilizing a manual technique and the Akoya OPAL system.
Spiteri et al. (2026) studied this question.