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Confocal Microscopy: Theory and Applications for Cellular Signaling

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Calcium Signaling Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 937))

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Abstract

The development of confocal microscopy and the commercial availability of confocal microscopes have provided many laboratories with an extremely powerful approach to examine cellular structure and function. Allied with the development of suitable tools, it is now possible to interrogate a wide range of structural and functional aspects on both fixed and live cells. Here we describe the basic principles underlying confocal microscopy and provide methodological accounts of how it can be used to study aspects related particularly (but not exclusively) to the expression, activation, and regulation of signaling by G-protein-coupled receptors. Specifically we provide detailed protocols for examining: the cellular expression and distribution of proteins by immunocytochemistry; cytoplasmic and organelle Ca2+ signaling using fluorescent indicators; second messenger generation using fluorescently tagged biosensors; and ligand/receptor internalization using fluorescently tagged peptide agonists and receptors.

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Acknowledgements

The Wellcome Trust (grant 061050), BBSRC (Biotechnology and Biological Sciences Research Council; grants 91/C15897 and 01/A4/C/07909), GlaxoSmithKline, and the Medical Research Council (MRC) are thanked for financial support.

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Authors and Affiliations

  1. Department of Pharmacology, University of Cambridge, Cambridge, UK

    Stephen C. Tovey

  2. Division of Reproductive Health, Uinversity of Warwick, Coventry, UK

    Paul J. Brighton

  3. MRC Toxicology Unit, University of Leicester, Leicester, UK

    Edward T. W. Bampton

  4. Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK

    Yan Huang

  5. Department of Cardiovascular Sciences, Division of Anaesthesia, Critical Care, and Pain Management, Leicester Royal Infirmary, University of Leicester, Leicester, UK

    Gary B. Willars

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  1. Stephen C. Tovey
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  2. Paul J. Brighton
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  3. Edward T. W. Bampton
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  4. Yan Huang
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  5. Gary B. Willars
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Correspondence to Stephen C. Tovey .

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Editors and Affiliations

  1. , Department of Cardiovascular Sciences, Leicester Royal Infirmary, Infirmary Square, Leicester, LE1 5WW, Leicestershire, United Kingdom

    David G. Lambert

  2. , Department of Cardiovascular Sciences, Leicester Royal Infirmary, Infirmary Square, Leicester, LE1 5WW, Leicestershire, United Kingdom

    Richard D. Rainbow

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Tovey, S.C., Brighton, P.J., Bampton, E.T.W., Huang, Y., Willars, G.B. (2013). Confocal Microscopy: Theory and Applications for Cellular Signaling. In: Lambert, D., Rainbow, R. (eds) Calcium Signaling Protocols. Methods in Molecular Biology, vol 937. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-086-1_3

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  • DOI: https://doi.org/10.1007/978-1-62703-086-1_3

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