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


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.


  1. Gerhardt, H. et al. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J. Cell Biol. 161, 1163–1177 (2003).

    Article  CAS  Google Scholar 

  2. Pitulescu, M.E., Schmidt, I., Benedito, R. & Adams, R.H. Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice. Nat. Protoc. 5, 1518–1534 (2010).

    Article  CAS  Google Scholar 

  3. Norrmén, C. et al. FOXC2 controls formation and maturation of lymphatic collecting vessels through cooperation with NFATc1. J. Cell Biol. 185, 439–457 (2009).

    Article  Google Scholar 

  4. Simons, M. et al. State-of-the-art methods for evaluation of angiogenesis and tissue vascularization: a scientific statement from the American Heart Association. Circ. Res. 116, e99–132 (2015).

    Article  CAS  Google Scholar 

  5. Zheng, W., Aspelund, A. & Alitalo, K. Lymphangiogenic factors, mechanisms, and applications. J. Clin. Invest. 124, 878–887 (2014).

    Article  CAS  Google Scholar 

  6. Potente, M., Gerhardt, H. & Carmeliet, P. Basic and therapeutic aspects of angiogenesis. Cell 146, 873–887 (2011).

    Article  CAS  Google Scholar 

  7. Garrett, W.S., Gordon, J.I. & Glimcher, L.H. Homeostasis and inflammation in the intestine. Cell 140, 859–870 (2010).

    Article  CAS  Google Scholar 

  8. Kamba, T. et al. VEGF-dependent plasticity of fenestrated capillaries in the normal adult microvasculature. Am. J. Physiol. Heart Circ. Physiol. 290, H560–H576 (2006).

    Article  CAS  Google Scholar 

  9. Yang, Y. et al. Anti-VEGF- and anti-VEGF receptor-induced vascular alteration in mouse healthy tissues. Proc. Natl. Acad. Sci. USA 110, 12018–12023 (2013).

    Article  CAS  Google Scholar 

  10. Stappenbeck, T.S., Hooper, L.V. & Gordon, J.I. Developmental regulation of intestinal angiogenesis by indigenous microbes via Paneth cells. Proc. Natl. Acad. Sci. USA 99, 15451–15455 (2002).

    Article  CAS  Google Scholar 

  11. Reinhardt, C. et al. Tissue factor and PAR1 promote microbiota-induced intestinal vascular remodelling. Nature 483, 627–631 (2012).

    Article  CAS  Google Scholar 

  12. Choe, K. et al. Intravital imaging of intestinal lacteals unveils lipid drainage through contractility. J. Clin. Invest. 125, 4042–4052 (2015).

    Article  Google Scholar 

  13. Bernier-Latmani, J. et al. DLL4 promotes continuous adult intestinal lacteal regeneration and dietary fat transport. J. Clin. Invest. 125, 4572–4586 (2015).

    Article  Google Scholar 

  14. Nurmi, H. et al. VEGF-C is required for intestinal lymphatic vessel maintenance and lipid absorption. EMBO Mol. Med. 7, 1418–1425 (2015).

    Article  CAS  Google Scholar 

  15. Petrova, T.V. et al. Defective valves and abnormal mural cell recruitment underlie lymphatic vascular failure in lymphedema distichiasis. Nat. Med. 10, 974–981 (2004).

    Article  CAS  Google Scholar 

  16. Sabine, A. et al. Mechanotransduction, PROX1, and FOXC2 cooperate to control Connexin37 and Calcineurin during lymphatic-valve formation. Dev. Cell 22, 430–445 (2012).

    Article  CAS  Google Scholar 

  17. Sabine, A. et al. FOXC2 and fluid shear stress stabilize postnatal lymphatic vasculature. J. Clin. Invest. 125, 3861–3877 (2015).

    Article  Google Scholar 

  18. Bazigou, E. et al. Integrin-alpha 9 is required for fibronectin matrix assembly during lymphatic valve morphogenesis. Dev. Cell 17, 175–186 (2009).

    Article  CAS  Google Scholar 

  19. Tatin, F. et al. Planar cell polarity protein Celsr1 regulates endothelial adherens junctions and directed cell rearrangements during valve morphogenesis. Dev. Cell 26, 31–44 (2013).

    Article  CAS  Google Scholar 

  20. Costa, M., Buffa, R., Furness, J.B. & Solcia, E. Immunohistochemical localization of polypeptides in peripheral autonomic nerves using whole mount preparations. Histochemistry 65, 157–165 (1980).

    Article  CAS  Google Scholar 

  21. Gabella, G. Neuron size and number in the myenteric plexus of the newborn and adult rat. J. Anat. 109, 81–95 (1971).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Fu, Y.-Y., Peng, S.-J., Lin, H.-Y., Pasricha, P.J. & Tang, S.-C. 3-D imaging and illustration of mouse intestinal neurovascular complex. Am. J. Physiol. Gastrointest. Liver Physiol. 304, G1–G11 (2013).

    Article  CAS  Google Scholar 

  23. Appleton, P.L., Quyn, A.J., Swift, S. & Näthke, I. Preparation of wholemount mouse intestine for high-resolution three-dimensional imaging using two-photon microscopy. J. Microsc. 234, 196–204 (2009).

    Article  CAS  Google Scholar 

  24. Moolenbeek, C. & Ruitenberg, E.J. The 'Swiss roll': a simple technique for histological studies of the rodent intestine. Lab. Anim. 15, 57–59 (1981).

    Article  CAS  Google Scholar 

  25. Gage, G.J., Kipke, D.R. & Shain, W. Whole animal perfusion fixation for rodents. J. Vis. Exp. 65, e3564, (2012).

    Google Scholar 

  26. Claxton, S. et al. Efficient, inducible Cre-recombinase activation in vascular endothelium. Genesis 46, 74–80 (2008).

    Article  CAS  Google Scholar 

  27. Zarkada, G., Heinolainen, K., Makinen, T., Kubota, Y. & Alitalo, K. VEGFR3 does not sustain retinal angiogenesis without VEGFR2. Proc. Natl. Acad. Sci. USA 112, 761–766 (2015).

    Article  CAS  Google Scholar 

  28. Benson, A.K. et al. Individuality in gut microbiota composition is a complex polygenic trait shaped by multiple environmental and host genetic factors. Proc. Natl. Acad. Sci. USA 107, 18933–18938 (2010).

    Article  CAS  Google Scholar 

  29. Shayan, R. et al. A system for quantifying the patterning of the lymphatic vasculature. Growth Factors 25, 417–425 (2007).

    Article  CAS  Google Scholar 

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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.

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Authors and Affiliations



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).

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