Abstract
Tissues are dynamic and complex biological systems composed of specialized cell types that interact with each other for proper biological function. To comprehensively characterize and understand the cell circuitry underlying biological processes within tissues, it is crucial to preserve their spatial information. Here we report a simple mounting technique to maximize the area of the tissue to be analyzed, encompassing the whole length of the murine gastrointestinal (GI) tract, from mouth to rectum. Using this method, analysis of the whole murine GI tract can be performed in a single slide not only by means of histological staining, immunohistochemistry and in situ hybridization but also by multiplexed antibody staining and spatial transcriptomic approaches. We demonstrate the utility of our method in generating a comprehensive gene and protein expression profile of the whole GI tract by combining the versatile tissue-rolling technique with a cutting-edge transcriptomics method (Visium) and two cutting-edge proteomics methods (ChipCytometry and CODEX-PhenoCycler) in a systematic and easy-to-follow step-by-step procedure. The entire process, including tissue rolling, processing and sectioning, can be achieved within 2–3 d for all three methods. For Visium spatial transcriptomics, an additional 2 d are needed, whereas for spatial proteomics assays (ChipCytometry and CODEX-PhenoCycler), another 3–4 d might be considered. The whole process can be accomplished by researchers with skills in performing murine surgery, and standard histological and molecular biology methods.
Key points
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In this protocol, a simple and versatile ‘Swiss rolling’ technique allows transcriptomic and proteomic profiling of the whole murine gastrointestinal tract with high spatial resolution.
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The method can be easily adapted to study changes in gene and/or protein expression under different environmental stimuli and genetic alterations, as well as to study tumor progression and variations in luminal microbiome in the gastrointestinal tract.
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Data availability
Datasets from this study that have been previously published are accessible via the corresponding accession number GSE169749, which includes 10x Visium spatial transcriptomics data of the murine colon in both steady state and during the recovery phase after dextran sodium sulfate colitis, as sourced from our primary publication12. The spatial transcriptomics data pertaining to the SI and ORES rolls, as referenced in this protocol (Fig. 6), constitute part of an unpublished dataset, and will be made publicly available concurrent with the publication of the primary articles. Figures 7 and 8 include raw data obtained from CODEX PhenoCycler and ChipCytometry analyses, and Fig. 9 includes raw data from optimization experiments for the development of ORES rolls. These data are available upon request. Source data are provided with this paper.
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Acknowledgements
We thank members of the Villablanca laboratory for helpful comments. G.M. and S.D. were supported by Cancerfonden (22 0513 and CAN 2016/1206, respectively). G.M. was supported by The Osteology Foundation (20-184). R.A.M was supported by ANID Becas Chile (74200049). S.D. was supported by Åke Weibergs Stiftelse (M21-0074 and M22-0048). A.S. and D.A.B were supported by the Deutsche Forschungsgemeinschaft (DFG; German Research Foundation) under Germany’s Excellence Strategy—EXC2151—390873048 and the SFB 1454. L.L. was supported by the Helmsley Foundation (1903-03785). E.J.V. was supported by grants from the Swedish Research Council, Vetenskapsrådet (VR) (grants 2018-02533 and 2021-01277), Formas (grant 2022-01066), Cancerfonden (22 2060 Pj) and the Wallenberg Academy Fellow program (2019.0315). Some schematics were partially created with BioRender.com.
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G.M., R.A.M., D.A.B., X.M.A. and S.D. performed the experiments and analysis. E.J.V. and G.M. conceived the idea. D.A.B. and A.S. provided CODEX data. J.F. and L.L. performed the bioinformatic analyses. G.M., R.A.M., S.D. and E.J.V. wrote the paper. All authors discussed the data, read and approved the manuscript.
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E.J.V. received research grants from F. Hoffmann–La Roche and is founder of Papervids.
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Nature Protocols thanks Daniel Mucida and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references using this protocol
Parigi, S. M. et al. Nat. Commun. 13, 828 (2022): https://doi.org/10.1038/s41467-022-28497-0
Frede, A. et al. Immunity 55, 2336–2351.e12 (2022): https://doi.org/10.1016/j.immuni.2022.11.002
Supplementary information
Supplementary Video 1
A comprehensive demonstration of the essential procedures involved in tissue isolation, processing, and mounting. Each critical step is depicted in detail to ensure clarity and to facilitate the replication of the methods presented in this protocol.
Source data
Source Data Fig. 8
Source data from CellScape quantitative analyses of ORES rolls plotted in Fig. 8.
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Monasterio, G., Morales, R.A., Bejarano, D.A. et al. A versatile tissue-rolling technique for spatial-omics analyses of the entire murine gastrointestinal tract. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-01001-2
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DOI: https://doi.org/10.1038/s41596-024-01001-2
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