Slide preparation for single-cell–resolution imaging of fluorescent proteins in their three-dimensional near-native environment

Abstract

In recent years, many mouse models have been developed to mark and trace the fate of adult cell populations using fluorescent proteins. High-resolution visualization of such fluorescent markers in their physiological setting is thus an important aspect of adult stem cell research. Here we describe a protocol to produce sections (150–200 μm) of near-native tissue with optimal tissue and cellular morphology by avoiding artifacts inherent in standard freezing or embedding procedures. The activity of genetically expressed fluorescent proteins is maintained, thereby enabling high-resolution three-dimensional (3D) reconstructions of fluorescent structures in virtually all types of tissues. The procedure allows immunofluorescence labeling of proteins to depths up to 50 μm, as well as a chemical 'Click-iT' reaction to detect DNA-intercalating analogs such as ethynyl deoxyuridine (EdU). Generation of near-native sections ready for imaging analysis takes approximately 2–3 h. Postsectioning processes, such as antibody labeling or EdU detection, take up to 10 h.

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Figure 1: Overview of the technique.
Figure 2: A versatile technique for tissues from mice and humans.
Figure 3: Optimal tissue morphology in sections of near-native tissue.
Figure 4: EdU labeling in near-native tissue sections.
Figure 5: Near-native tissue sections with elevated background levels.

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Acknowledgements

We thank J. Kuipers and the Hubrecht Imaging Center for their support.

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Authors

Contributions

H.J.S. and A.G.S. conducted experiments and wrote the manuscript, G.D. and P.D.S. provided materials, and H.C. wrote the manuscript.

Corresponding author

Correspondence to Hans Clevers.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

Embedding tissue in LMA, Procedure Steps 4-7. (MOV 23985 kb)

Supplementary Video 2

Sectioning LMA-embedded tissue and mounting near-native sections, Procedure Steps 7-12, 14 and 15. (MOV 24883 kb)

Supplementary Table 1

Antibodies successfully used for staining near-native tissue sections. (XLSX 13 kb)

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Snippert, H., Schepers, A., Delconte, G. et al. Slide preparation for single-cell–resolution imaging of fluorescent proteins in their three-dimensional near-native environment. Nat Protoc 6, 1221–1228 (2011). https://doi.org/10.1038/nprot.2011.365

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