Spatial Transcriptomics of Early Tooth Morphogenesis in Formalin-fixed Paraffin-embedded Mouse Embryonic Tissue.

The developing tooth comprises diverse and highly specialized cell populations that work together to maintain proper form and function. Elucidating the interactions among these cells and their surrounding microenvironment is critical for understanding the regulatory mechanisms underlying normal tooth development. Perturbations in these processes can result in congenital disorders such as tooth agenesis, dentinogenesis imperfecta, and amelogenesis imperfecta. Despite the substantial progress enabled by single-cell RNA sequencing (scRNA-seq) in revealing cellular heterogeneity, it does not preserve the spatial context of cells within tissues, limiting the ability to relate gene expression to tissue architecture. Spatial transcriptomic technologies address this limitation by integrating high-resolution gene expression profiling with the preservation of native tissue architecture, enabling the in situ localization of molecular signatures. Here, we describe a step-by-step protocol for the collection, fixation, and paraffin embedding of mouse embryonic craniofacial tissue suitable for downstream spatial transcriptomic applications. The workflow details optimized sectioning and handling of formalin-fixed, paraffin-embedded tissue to preserve RNA integrity and tissue morphology for high-resolution spatial analysis. This method is compatible with sequencing and image-based spatial transcriptomics platforms, enabling reproducible spatial transcriptomic profiling of early tooth morphogenesis in mouse embryos. This approach offers powerful insights into the spatial organization and functional dynamics of craniofacial structures in both developmental and pathological states, providing a critical framework for linking molecular mechanisms to tissue morphology.