Correlative light and electron microscopy reveals discrepancy between gold and fluorescence labelling.

Autor: VAN Elsland DM; Division of Bio-organic Synthesis, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Leiden, The Netherlands.; Institute for Chemical Immunology, Gorlaeus Laboratories, Leiden University, Leiden, The Netherlands., Bos E; Department of Molecular Cell Biology, Section Electron Microscopy, Leiden University Medical Center, Leiden, The Netherlands., Pawlak JB; Division of Bio-organic Synthesis, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Leiden, The Netherlands.; Institute for Chemical Immunology, Gorlaeus Laboratories, Leiden University, Leiden, The Netherlands., Overkleeft HS; Division of Bio-organic Synthesis, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Leiden, The Netherlands.; Institute for Chemical Immunology, Gorlaeus Laboratories, Leiden University, Leiden, The Netherlands., Koster AJ; Department of Molecular Cell Biology, Section Electron Microscopy, Leiden University Medical Center, Leiden, The Netherlands., VAN Kasteren SI; Division of Bio-organic Synthesis, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Leiden, The Netherlands.; Institute for Chemical Immunology, Gorlaeus Laboratories, Leiden University, Leiden, The Netherlands.
Jazyk: angličtina
Zdroj: Journal of microscopy [J Microsc] 2017 Sep; Vol. 267 (3), pp. 309-317. Date of Electronic Publication: 2017 May 04.
DOI: 10.1111/jmi.12567
Abstrakt: Electron microscopy (EM) is traditionally employed as a follow-up to fluorescence microscopy (FM) to resolve the cellular ultrastructures wherein fluorescently labelled biomolecules reside. In order to translate the information derived from FM studies to EM analysis, biomolecules of interest must be identified in a manner compatible with EM. Although fluorescent signals can serve this purpose when FM is combined with EM in correlative light and electron microscopy (CLEM), the traditional immunogold labelling remains commonly used in this context. In order to investigate how much these two strategies relate, we have directly compared the subcellular localization of on-section fluorescence labelling with on-section immunogold labelling. In addition to antibody labelling of LAMP-1, bioorthogonal click labelling was used to localize soluble cysteine cathepsins or membrane-associated sialylated glycans. We reveal and characterize the existence of inherent discrepancies between the fluorescence signal and the distribution of gold particles in particular in the case of membrane-associated antigens.
(© 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.)
Databáze: MEDLINE
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