Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Immunocytochemical techniques for whole-mount in situ protein localization in plants

Abstract

As the field of plant molecular biology is swiftly advancing, a need has been created for methods that allow rapid and reliable in situ localization of proteins in plant cells. Here we describe a whole-mount 'immunolocalization' technique for various plant tissues, including roots, hypocotyls, cotyledons, young primary leaves and embryos of Arabidopsis thaliana and other species. The detailed protocol, recommended controls and troubleshooting are presented, along with examples of applications. The protocol consists of five main procedures: tissue fixation, tissue permeation, blocking, primary and secondary antibody incubation. Notably, the first procedure (tissue fixation) includes several steps (4–12) that are absolutely necessary for protein localization in hypocotyls, cotyledons and young primary leaves but should be omitted for other tissues. The protocol is usually done in 3 days, but could also be completed in 2 days.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Examples of whole-mount immunolocalization of various proteins in different plant tissues.

References

  1. Brandizzi, F., Fricker, M. & Hawes, C. A greener world: The revolution in plant bioimaging. Nat. Rev. Mol. Cell Biol. 7, 520–530 (2002).

    Article  Google Scholar 

  2. Lauber, M.H. et al. The Arabidopsis KNOLLE protein is a cytokinesis-specific syntaxin. J. Cell Biol. 139, 1485–1493 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Friml, J., Benková, E., Mayer, U., Palme, K. & Muster, G. Automated whole-mount localization techniques for plant seedlings. Plant J. 34, 115–124 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Paciorek, T., Sauer, M., Balla, J., Wisniewska, J. & Friml, J. Immunocytochemical technique for protein localization in sections of plant tissues. Nat. Protocols doi:10.1038/nprot.2006.16.

    Article  CAS  PubMed  Google Scholar 

  5. Pimpl, P., Movafeghi, A ., Coughlan, S., Denecke, J., Hilmer, S. & Robinson, D.G. In situ localization and in vitro induction of plant COPI-coated vesicles. Plant Cell 11, 2213–2236 (2000).

    Google Scholar 

  6. Paciorek, T. et al. Auxin inhibits endocytosis and promotes its own efflux from cells. Nature 435, 1251–1256 (2005).

    Article  CAS  Google Scholar 

  7. Swarup, R. et al. Structure-function analysis of the presumptive Arabidopsis auxin permease AUX1. Plant Cell 16, 3069–3083 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to C. Luschnig, W. Michalke, D. Robinson and R. Swarup for sharing material, and we thank P. Brewer for critical reading of the manuscript and discussions. M.S. and J.F. were supported by VolkswagenStiftung and the European Molecular Biology Organization Young Investigator Program; T.P. was supported by the Deutsche Forschungsgemeinschaft (SFB 446); and E.B. was supported by the Margarete von Wrangell-Habilitationsprogramm.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Jiří Friml.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Sauer, M., Paciorek, T., Benková, E. et al. Immunocytochemical techniques for whole-mount in situ protein localization in plants. Nat Protoc 1, 98–103 (2006). https://doi.org/10.1038/nprot.2006.15

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.15

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing