文章基本信息

标题：A highly sensitive protocol for microscopy of alkyne lipids and fluorescently tagged or immunostained proteins
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作者：Anne Gaebler ; Anke Penno ; Lars Kuerschner 等
期刊名称：JLR Papers In Press
印刷版ISSN：0022-2275
电子版ISSN：1539-7262
出版年度：2016
卷号：57
期号：10
页码：1934-1947
DOI：10.1194/jlr.D070565
语种：English
出版社：American Society for Biochemistry and Molecular Biology
摘要：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.
关键词：click chemistry ; lipid imaging ; copper-catalyzed azide-alkyne cycloaddition ; immunohistochemistry ; fluorescent proteins ; fluorescence microscopy ; molecular imaging ; chemical synthesis ; lipid biochemistry