Protocol for high-plex, whole-slide imaging of human formalin-fixed paraffin-embedded tissue using PhenoCycler-Fusion
Meg L. Donovan, Niyati Jhaveri, Ning Ma, Bassem Ben Cheikh, James DeRosa, Ritu Mihani, Naomi Berrell, Jacky Y. Suen, James Monkman, John F. Fraser, Arutha Kulasinghe
Abstract
Single-cell spatial analysis of proteins is rapidly becoming increasingly important in revealing biological insights. Here, we present a protocol for automated high-plex multi-slide immunofluorescence staining and imaging of human head and neck cancer formalin-fixed paraffin-embedded (FFPE) sections using PhenoCycler-Fusion 2.0 technology. We describe steps for preparing human head and neck cancer FFPE tissues, staining with a panel of immunophenotyping markers, and Flow Cell assembly. We then detail procedures for setting up for a PhenoCycler-Fusion run, post-run Flow Cell removal, and downstream analyses. For complete details on the use and execution of this protocol, please refer to Jhaveri et al. 1 • Protocol for preparing and staining human FFPE tissue slides for PhenoCycler-Fusion • Flow Cell assembly and preparation for a PhenoCycler-Fusion run • Steps for post-PhenoCycler-Fusion run Flow Cell removal for same-slide H&E staining • Example analysis workflow for cell segmentation, phenotyping, and spatial analyses Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Single-cell spatial analysis of proteins is rapidly becoming increasingly important in revealing biological insights. Here, we present a protocol for automated high-plex multi-slide immunofluorescence staining and imaging of human head and neck cancer FFPE sections using PhenoCycler-Fusion 2.0 technology. We describe steps for preparing human head and neck cancer FFPE tissues, staining with a panel of immunophenotyping markers, and Flow Cell assembly. We then detail procedures for setting up for a PhenoCycler-Fusion run, post-run Flow Cell removal, and downstream analyses.