Abstract
Abstract The murine embryo model is one of the most common animal models for studying developmental biology. Embryonic development is a highly regulated process with spatial and temporal expression of proteins for regional specialization and tissue-specific functions. Application of spatial proteomics to developing embryos enables us to understand cellular organization and interactions within tissues along with regulatory mechanisms at the single-cell level. In this study, we applied a 36-plex panel, specifically designed for analysis of mouse embryos at different stages of development using the PhenoCycler™-Fusion (PCF) 2.0 Platform. This panel was developed by selecting biomarkers for immune cell lineages, immune activation, proliferation, structural and neuronal components. Purified antibodies were tagged to PhenoCycler barcodes and verified for specificity and reproducibility. After successful validation, the panel was deployed on the FFPE mouse embryos and ultrahigh-plex whole-slide imaging was performed using the PCF 2.0 platform. Unsupervised clustering analysis and deep phenotyping were performed to generate a high-resolution spatial atlas of mouse embryos at different stages of development. The tools developed as part of this study can be used to conduct in-depth research on various aspects of organogenesis and to study the spatio-temporal dynamics of cellular and molecular expression in developing embryos.
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