title: Localization of fluorescently tagged protein to plasmodesmata by correlative light and electron microscopy.
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.
authors.affiliation: Delaware Biotechnology Institute, University of Delaware, Delaware Technology Park, 15 Innovation Way, Newark, DE, 19711, USA.
authors.forename: Shannon
authors.initials: S
authors.lastname: Modla
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Caplan
authors.forename: Kirk J
authors.initials: KJ
authors.lastname: Czymmek
authors.forename: Jung-Youn
authors.initials: JY
authors.lastname: Lee
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000648:ultrastructure*; D029681:Arabidopsis Proteins / Q000032:analysis* / Q000235:genetics; D001692:Biological Transport; D002473:Cell Wall / Q000235:genetics / Q000648:ultrastructure*; D005404:Fixatives / Q000737:chemistry; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics; D046529:Microscopy, Electron, Transmission / Q000706:statistics & numerical data; D008856:Microscopy, Fluorescence / Q000706:statistics & numerical data; D008867:Microtomy; D030821:Plants, Genetically Modified; D031425:Plasmodesmata / Q000235:genetics / Q000648:ultrastructure*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D036226:Seedlings / Q000235:genetics / Q000648:ultrastructure; D016707:Tissue Fixation
chemical_list: D029681:Arabidopsis Proteins; D005404:Fixatives; D008565:Membrane Proteins; C586778:PDLP5 protein, Arabidopsis; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins