814.1 Multiplexed Immunofluorescence Phenocycler-Fusion® Imaging of FFPE Lung Sections Following Mass Spectrometry V1

This protocol describes multiplexed immunofluorescent (MxIF) staining and imaging of FFPE lung tissue sections utilizing the Phenocycler-Fusion platform (Akoya Biosciences) following spatial analysis of N-Glycans with MALDI-MSI. The approach for MxIF is based on the CODEX multiplexed immunofluorescence staining technology developed by Garry Nolan and colleagues (1). The protocol is closely aligned with the attached Phenocycler-Fusion User Guide provided by Akoya (2). The protocol includes A) FFPE lung tissue section preparation, B) Reference to spatial N-glycomics with MALDI-MSI for human lung tissue C) Labeling of lung tissue sections with antibody-barcode conjugates, D) Barcode-Reporter plate and experiment design, E) Multiplexed imaging and analysis, F) Reference for H&E stain to follow MxIF and G) Reference to custom antibody conjugation. Thus, the protocol is designed to provide information regarding specific reagents (i.e., antibodies), conditions (i.e., dilutions), and procedures used in multiplexed immunofluorescent staining of lung tissue.