Roy Lardenoije

This is a protocol for preparing fresh-frozen kidney tissue for in situ spatial transcriptomics using a custom probe panel with the 10X Xenium Analyzer platform. Slide preparation is a multi-day process encompassing cryosectioning, probe hybridization, subsequent ligation and amplification, quenching and nuclei staining, and in situ imaging. Each phase of the workflow is extensively detailed within published 10X User Guides, which are cited throughout this version. After data is collected, slides are H&E stained for histopathological evaluation of healthy and diseased tissue, and used for integrated data registration to neighboring images and modalities. There are sample evaluation and QC measures built into the 10X protocols, which are carefully observed in addition to our own go/no-go criteria.

Summary: Spatial ’omics technologies are a powerful tool for mapping the relationship between cellular organization and molecular distributions in healthy and diseased tissue microenvironments. Here, we describe a novel multimodal pipeline that represents experimental and computational advances for spatiomolecular analysis of tissue samples across molecular classes. This adaptable method integrates matrix-assisted laser desorption/ionization imaging mass spectrometry spatial lipidomics, spatial transcriptomics, protein imaging via multiplexed immunofluorescence microscopy, and histopathological staining to uncover spatiomolecular profiles associated with unique cellular niches and pathological features. We demonstrate the power of this approach using two different complex human disease systems: Alzheimer’s disease in human brain tissue and type 2 diabetes mellitus in the human pancreas. This work establishes and demonstrates a generalizable framework for multimodal spatial integration, enabling precise mapping of molecular mechanisms that underlie complex tissue pathologies.

Recent studies have demonstrated that the dorsal raphe nucleus (DRN) is among the first brain regions affected in Alzheimer’s disease. Hence, in this study we conducted the first comprehensive epigenetic analysis of the DRN in AD, targeting both bulk tissue and single isolated cells. The Illumina Infinium MethylationEPIC BeadChip array was used to analyze the bulk tissue, assessing differentially modified positions (DMoPs) and regions (DMoRs) associated with Braak stage. The strongest Braak stage-associated DMoR in TNXB was targeted in a second patient cohort utilizing single laser-capture microdissected serotonin-positive (5-HT+) and -negative (5-HT-) cells isolated from the DRN. Our study revealed previously identified epigenetic loci, including TNXB and PGLYRP1 , and novel loci, including RBMXL2 , CAST , GNAT1 , MALAT1 , and DNAJB13 . Strikingly, we found that the methylation profile of TNXB depends both on disease phenotype and cell type analyzed, emphasizing the significance of single cell(-type) neuroepigenetic studies in AD.