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Root Development pp 131–143Cite as

Optimized Whole-Mount In Situ Immunolocalization for Arabidopsis thaliana Root Meristems and Lateral Root Primordia

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1761))

Abstract

Immunolocalization is a valuable tool for cell biology research that allows to rapidly determine the localization and expression levels of endogenous proteins. In plants, whole-mount in situ immunolocalization remains a challenging method, especially in tissues protected by waxy layers and complex cell wall carbohydrates. Here, we present a robust method for whole-mount in situ immunolocalization in primary root meristems and lateral root primordia in Arabidopsis thaliana. For good epitope preservation, fixation is done in an alkaline paraformaldehyde/glutaraldehyde mixture. This fixative is suitable for detecting a wide range of proteins, including integral transmembrane proteins and proteins peripherally attached to the plasma membrane. From initiation until emergence from the primary root, lateral root primordia are surrounded by several layers of differentiated tissues with a complex cell wall composition that interferes with the efficient penetration of all buffers. Therefore, immunolocalization in early lateral root primordia requires a modified method, including a strong solvent treatment for removal of hydrophobic barriers and a specific cocktail of cell wall-degrading enzymes. The presented method allows for easy, reliable, and high-quality in situ detection of the subcellular localization of endogenous proteins in primary and lateral root meristems without the need of time-consuming crosses or making translational fusions to fluorescent proteins.

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Author information

Authors and Affiliations

  1. Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Barcelona, Spain

    Michael Karampelias

  2. Facultad de Recursos Naturales Renovables, Universidad Arturo Prat, Iquique, Chile

    Ricardo Tejos

  3. Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria

    Jiří Friml

  4. Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium

    Steffen Vanneste

  5. VIB Center for Plant Systems Biology, Ghent, Belgium

    Steffen Vanneste

Authors
  1. Michael Karampelias
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  2. Ricardo Tejos
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  3. Jiří Friml
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  4. Steffen Vanneste
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Corresponding author

Correspondence to Steffen Vanneste .

Editor information

Editors and Affiliations

  1. Gregor Mendel Institute of Molecular Plant Biology, Vienna, Wien, Austria

    Daniela Ristova

  2. Institute of Applied Genetics & Cell Biology, University of Natural Resources and Life Sciences, Vienna, Wien, Austria

    Elke Barbez

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Karampelias, M., Tejos, R., Friml, J., Vanneste, S. (2018). Optimized Whole-Mount In Situ Immunolocalization for Arabidopsis thaliana Root Meristems and Lateral Root Primordia. In: Ristova, D., Barbez, E. (eds) Root Development. Methods in Molecular Biology, vol 1761. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7747-5_10

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  • DOI: https://doi.org/10.1007/978-1-4939-7747-5_10

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7746-8

  • Online ISBN: 978-1-4939-7747-5

  • eBook Packages: Springer Protocols

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