DNA-barcode-based Multiplex Immunofluorescence Imaging to Analyze FFPE Specimens from Genetically Reprogrammed Murine Melanoma

Presented here is an emerging DNA-barcode-based multiplex imaging technique based on Co-Detection-by-indEXing that analyzes the spatial proteomics of tissue microenvironments. Successful imaging requires a repertoire of well-designed and properly validated antibody panels, but very few currently exist for formalin-fixed paraffin-embedded (FFPE) samples. FFPE offers several advantages over fresh-frozen specimens, such as widespread availability, ease of handling and storage, and the ability to make tissue microarrays (TMAs). Here, we present a protocol to develop an antibody panel for visualizing and analyzing FFPE tissues from a murine melanoma model treated with nanoparticles, which deliver plasmid DNA encoding immunologic signals for tumor microenvironment reprogramming. We also describe an image analysis pipeline using open-source computational tools for annotating tissues, segmenting cells, processing proteomics data, phenotyping cell populations, and quantifying spatial metrics. The protocol offers applications for designing antibody panels in murine FFPE and generating novel insights into the spatial proteomics of complex tissue microenvironments.