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Optimized whole-mount in situ immunolocalization for Arabidopsis thaliana root meristems and lateral root primordia

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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.
Keywords
Arabidopsis, In situ immunolocalization, Lateral root primordia

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MLA
Karampelias, Michael, Ricardo Tejos Ulloa, Jiří Friml, et al. “Optimized Whole-mount in Situ Immunolocalization for Arabidopsis Thaliana Root Meristems and Lateral Root Primordia.” Root Development : Methods and Protocols. Ed. Daniela Ristova & Elke Barbez. Vol. 1761. New York, NY, USA: Springer Humana Press, 2018. 131–143. Print.
APA
Karampelias, Michael, Tejos Ulloa, R., Friml, J., & Vanneste, S. (2018). Optimized whole-mount in situ immunolocalization for Arabidopsis thaliana root meristems and lateral root primordia. In D. Ristova & E. Barbez (Eds.), Root development : methods and protocols (Vol. 1761, pp. 131–143). New York, NY, USA: Springer Humana Press.
Chicago author-date
Karampelias, Michael, Ricardo Tejos Ulloa, Jiří Friml, and Steffen Vanneste. 2018. “Optimized Whole-mount in Situ Immunolocalization for Arabidopsis Thaliana Root Meristems and Lateral Root Primordia.” In Root Development : Methods and Protocols, ed. Daniela Ristova and Elke Barbez, 1761:131–143. New York, NY, USA: Springer Humana Press.
Chicago author-date (all authors)
Karampelias, Michael, Ricardo Tejos Ulloa, Jiří Friml, and Steffen Vanneste. 2018. “Optimized Whole-mount in Situ Immunolocalization for Arabidopsis Thaliana Root Meristems and Lateral Root Primordia.” In Root Development : Methods and Protocols, ed. Daniela Ristova and Elke Barbez, 1761:131–143. New York, NY, USA: Springer Humana Press.
Vancouver
1.
Karampelias M, Tejos Ulloa R, Friml J, Vanneste S. Optimized whole-mount in situ immunolocalization for Arabidopsis thaliana root meristems and lateral root primordia. In: Ristova D, Barbez E, editors. Root development : methods and protocols. New York, NY, USA: Springer Humana Press; 2018. p. 131–43.
IEEE
[1]
M. Karampelias, R. Tejos Ulloa, J. Friml, and S. Vanneste, “Optimized whole-mount in situ immunolocalization for Arabidopsis thaliana root meristems and lateral root primordia,” in Root development : methods and protocols, vol. 1761, D. Ristova and E. Barbez, Eds. New York, NY, USA: Springer Humana Press, 2018, pp. 131–143.
@incollection{8558104,
  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.},
  author       = {Karampelias, Michael and Tejos Ulloa, Ricardo and Friml, Jiří and Vanneste, Steffen},
  booktitle    = {Root development : methods and protocols},
  editor       = {Ristova, Daniela and Barbez, Elke},
  isbn         = {9781493977468},
  issn         = {1064-3745},
  keywords     = {Arabidopsis,In situ immunolocalization,Lateral root primordia},
  language     = {eng},
  pages        = {131--143},
  publisher    = {Springer Humana Press},
  series       = {Methods in Molecular Biology},
  title        = {Optimized whole-mount in situ immunolocalization for Arabidopsis thaliana root meristems and lateral root primordia},
  url          = {http://dx.doi.org/10.1007/978-1-4939-7747-5_10},
  volume       = {1761},
  year         = {2018},
}

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