Understanding the structure–function relationships between diverse cell types in a complex organ environment requires detailed in situ reconstruction of cell-associated molecular properties in the context of 3D, macro-scale tissue architecture. We recently developed clearing-enhanced 3D (Ce3D), a simple and effective method for tissue clearing that achieves excellent transparency; preserves cell morphology, tissue architecture, and reporter molecule fluorescence; and is robustly compatible with direct immunolabeling. These characteristics permit high-quality multiplex fluorescence microscopy of large tissue volumes, as well as image analysis using advanced platforms such as volumetric histocytometry, collectively allowing quantitative characterization of cells with respect to their spatial positioning within tissues on the basis of phenotypic and functional markers. Ce3D clearing is fast, achieving robust transparency of most tissues within 24 h, albeit still necessitating additional time for staining, imaging, and analysis (1–2 weeks). Here, we provide detailed procedures for tissue clearing using Ce3D, including optimized workflows for tissue processing and staining, as well as treatment of difficult-to-clear organs such as the brain. We also describe a new procedure for RNA detection in Ce3D-treated tissues, as well as provide additional details for the volumetric histocytometry data processing steps. Finally, we discuss limitations and work-around strategies for improving antibody-based tissue immunolabeling, fluorophore multiplexing, large-volume microscopy, and computational analysis of large image datasets. Together, these detailed procedures and solutions for high-resolution volumetric microscopy with Ce3D enable quantitative visualization of cells and tissues at a high level of detail, allowing exploration of cellular spatial relationships in a variety of tissue settings.
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The image datasets generated and/or analyzed during the current study are available from the corresponding authors upon reasonable request.
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We thank K. Mao for helping with gut and mammary gland tissue preparation. In addition, we thank all members of the Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases (NIAID) at the NIH for many helpful comments during the course of these experiments. This research was supported by the Intramural Research Program, NIAID, NIH, and NIH grant no. R01AI134713 MYG.
A patent for the methodology described in this paper was filed with the US Patent Office (PCT Patent Application PCT/US2017/049133, HHS reference no. E–168–2016, ‘Enhanced Tissue Clearing Solution, Clearing-Enhanced 3D (Ce3D), Compatible With Advanced Fluorescence Microscopy Imaging’).
Journal peer review information: Nature Protocols thanks Michael Donovan and Constantinos Petrovas for their contribution to the peer review of this work.
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Key references using this protocol
Li, W., Germain, R.N. and Gerner, M.Y. PNAS 114, E7321–E7330 (2017): https://www.pnas.org/content/114/35/E7321
Gerner, M.Y., Kastenmuller, W., Ifrim, I., Kabat, J. & Germain, R.N. Immunity 37, 364–376 (2012): https://www.cell.com/immunity/fulltext/S1074-7613(12)00321-4
Liu, Z. et al. Nature 528, 225–230 (2015): https://www.nature.com/articles/nature16169
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Li, W., Germain, R.N. & Gerner, M.Y. High-dimensional cell-level analysis of tissues with Ce3D multiplex volume imaging. Nat Protoc 14, 1708–1733 (2019). https://doi.org/10.1038/s41596-019-0156-4
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