title: A highly sensitive protocol for microscopy of alkyne lipids and fluorescently tagged or immunostained proteins.
abstract: The demand to study the cellular localization of specific lipids has led to recent advances in lipid probes and microscopy. Alkyne lipids bear a small, noninterfering tag and can be detected upon click reaction with an azide-coupled reporter. Fluorescent alkyne lipid imaging crucially depends on appropriate azide reporters and labeling protocols that allow for an efficient click reaction and therefore a sensitive detection. We synthesized several azide reporters with different spacer components and tested their suitability for alkyne lipid imaging in fixed cells. The implementation of a copper-chelating picolyl moiety into fluorescent or biotin-based azide reagents strongly increased the sensitivity of the imaging routine. We demonstrate the applicability and evaluate the performance of this approach using different lipid classes and experimental setups. As azide picolyl reporters allow for reduced copper catalyst concentrations, they also enable coimaging of alkyne lipids with multiple fluorescent proteins including enhanced green fluorescent protein. Alternatively, and as we also show, microscopy of alkyne lipids can be combined with protein detection by immunocytochemistry. In summary, we present a robust, sensitive, and highly versatile protocol for the labeling of alkyne lipids with azide-coupled reporters for fluorescence microscopy that can be combined with different protein detection and imaging techniques.
authors: Life & Medical Sciences Institute (LIMES), University of Bonn, D-53115 Bonn, Germany.
authors: Anne
authors: A
authors: Gaebler
authors: Life & Medical Sciences Institute (LIMES), University of Bonn, D-53115 Bonn, Germany.
authors: Anke
authors: A
authors: Penno
authors: Life & Medical Sciences Institute (LIMES), University of Bonn, D-53115 Bonn, Germany.
authors: Lars
authors: L
authors: Kuerschner
authors: Life & Medical Sciences Institute (LIMES), University of Bonn, D-53115 Bonn, Germany. Electronic address: cthiele@uni-bonn.de.
authors: Christoph
authors: C
authors: Thiele
mesh_terms: D045744:Cell Line, Tumor; D005456:Fluorescent Dyes / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D008055:Lipids; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: chemical synthesis; click chemistry; copper-catalyzed azide-alkyne cycloaddition; fluorescence microscopy; fluorescent proteins; immunohistochemistry; lipid biochemistry; lipid imaging; molecular imaging