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