We developed a method that utilizes floating mounting of thin sections of fixed frozen mouse lung tissue onto Xenium slides for the fluorescent in situ hybridization (FISH) and imaging –based spatial transcriptomics analysis of gene expression using the Xenium platform provided by 10X Genomics. Spatial transcriptomics techniques provide a comprehensive view by merging gene expression data with spatial context within their native tissue architecture. However, the Xenium pipeline has been validated only for formalin-fixed paraffin-embedded (FFPE) and fresh frozen sections by 10X Genomics. Notably, many researchers prefer paraformaldehyde-fixed cryosections for immunohistochemistry and in situ hybridization. In our study, we assessed the compatibility of standard fixed frozen mouse lung sections with the Xenium protocol. Our findings reveal that these sections not only align well with the Xenium platform but also offer superb imaging and gene expression quantification, even with limited number of genes in the Xenium panel. This protocol can serve as a valuable resource for preparing various tissues where FFPE and fresh frozen samples present challenges.

Xenium platform was used for the spatial transcriptomic analysis of human DRG neurons, 100 marker genes were selected as the customized probe panel and hybridized to fresh frozen hDRG sections. Manual segmentation of each neuron soma was performed, based on expressions of pan-neuronal marker gene PGP9.5, satellite glia cell marker FAB7B, and the corresponding H.E. staining. In total, 1340 neurons were identified (excluding 75 region-of-interest with poor or unclear neuronal soma morphology in H & E staining) and clustered into 16 groups. The 16 clusters were assigned as different cell types based on marker genes expression.

This repository contains (1) all Xenium outputs as .zip files, (2) CD31 immunohistochemistry (IHC) staining in .czi format and the (3) .rds files related to 3 tissue sections from 2 human donors analysed by Xenium. Xenium and IHC were performed in sequential tissue sections from the same blocks. These datasets belong to a larger study to understand vascular co-option in brain micro-metastasis (Pedro García-Gómez et al., 2024). Please visit https://github.com/....... to obtain further information about the code and analysis pipeline used.

This dataset is available through Zenodo for academic non-commercial use only. To request access, you must use your work/university email address and must submit the following details to ctc.peace@ucl.ac.uk:

Your Full Name, Role and Affiliated Institution

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Acknowledgement of the restrictions for academic non-commercial use only

Access Request will be reviewed by the Data Access Committee (ctc.peace@ucl.ac.uk)

The dataset can only be used by the applicants named on the application and for the intended use specified in the request. Copying or redistribution of this dataset is strictly prohibited.

Additional file 3: Table S3. Cardiomyocyte gene panel for Molecular Cartography experiment.