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High-resolution 3D analysis of mouse small-intestinal stroma

Abstract

Here we detail a protocol for whole-mount immunostaining of mouse small-intestinal villi that can be used to generate high-resolution 3D images of all gut cell types, including blood and lymphatic vessel cells, neurons, smooth muscle cells, fibroblasts and immune cells. The procedure describes perfusion, fixation, dissection, immunostaining, mounting, clearing, confocal imaging and quantification, using intestinal vasculature as an example. As intestinal epithelial cells prevent visualization with some antibodies, we also provide an optional protocol to remove these cells before fixation. In contrast to alternative current techniques, our protocol enables the entire villus to be visualized with increased spatial resolution of cell location, morphology and cell–cell interactions, thus allowing for easy quantification of phenotypes. The technique, which takes 7 d from mouse dissection to microscopic examination, will be useful for researchers who are interested in most aspects of intestinal biology, including mucosal immunology, infection, nutrition, cancer biology and intestinal microbiota.

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Figure 1: Whole-mount immunostaining allows high-resolution imaging of intestinal stroma.
Figure 2: All intestinal stromal cell types can be visualized with intestinal whole-mount immunostaining.
Figure 3: Experimental outline for preparing intestine for simultaneous imaging by paraffin sectioning, whole mount with epithelial cells (WM/EP+) and whole mount without epithelial cells (WM/EP).
Figure 4: Experimental details.
Figure 5: Expected results.

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Acknowledgements

We thank P. Baluk for useful discussions, S. Davanture and C. Beauverd for technical assistance, and C. Cisarovsky and C. Mauri for critical reading of the manuscript. The Animal, Cellular Imaging and Mouse Pathology Facilities of UNIL are gratefully acknowledged. This work was supported by Swiss National Science Foundation (PPP0033-114898, CRSII3-141811 and 31003A-156266) and Fondation MEDIC grants to T.V.P.

Author information

Authors and Affiliations

Authors

Contributions

J.B.-L. performed the experiments; and J.B.-L. and T.V.P. designed the protocol and wrote the manuscript.

Corresponding author

Correspondence to Tatiana V Petrova.

Integrated supplementary information

Supplementary Figure 1 Example of necessity for WM –EP protocol where strong epithelial staining prevents stroma visualization in most villi.

Whole-mount immunostaining of intestinal villi for Dll4 (cyan). Dll4 is highly expressed in intestinal epithelial cells preventing visualization of stromal Dll4 (outlined). Scale bar: 50 μm.

Supplementary information

Supplementary Information

Supplementary Figure 1, Supplementary Table 1 and Supplementary Methods (PDF 527 kb)

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Bernier-Latmani, J., Petrova, T. High-resolution 3D analysis of mouse small-intestinal stroma. Nat Protoc 11, 1617–1629 (2016). https://doi.org/10.1038/nprot.2016.092

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