Abstract
Plasmodesmata (PD) are intercellular communication channels that form long, membrane-lined cylinders across cellular junctions. A fluorescent-tagging approach is most commonly used for an initial assessment to address whether a protein of interest may localize or associate with PD domain. However, owing to the dimension of PD being at nanoscale, PD-associated fluorescent signals are detected only as small spots scattered at the cell periphery, hence requiring additional confirmatory evidence. Immunogold labeling provides such information, but suitable antibodies are not always available and morphological preservation is often compromised with this approach. Here we describe an alternative approach using a correlative light and electron microscopy (CLEM) technique, which combines fluorescent imaging and transmission electron microscopy. By employing this method, a clear correlation between fluorescent speckles and the presence of individual or clusters of PD is achieved.
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Acknowledgements
The research pertinent to the development of this protocol was supported by grants provided by the National Science Foundation (IOB 0954931) and partially by the grants from the National Center for Research Resources (5P30RR031160-03) and the National Institute of General Medical Sciences (8 P30 GM103519-03) from the National Institutes of Health to J.-Y. L.
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Modla, S., Caplan, J.L., Czymmek, K.J., Lee, JY. (2015). Localization of Fluorescently Tagged Protein to Plasmodesmata by Correlative Light and Electron Microscopy. In: Heinlein, M. (eds) Plasmodesmata. Methods in Molecular Biology, vol 1217. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1523-1_8
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DOI: https://doi.org/10.1007/978-1-4939-1523-1_8
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