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CLEM Methods for Studying Primary Cilia

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Cilia

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1454))

Abstract

CLEM (correlated light and electron microscope) imaging is a highly useful technique for examining primary cilia. With CLEM, it is possible to determine the distribution of tagged proteins along the ciliary membrane and axoneme with high precision. Scanning electron microscopy (SEM) permits measurement of ciliary length and orientation in relation to nearby cellular structures in a 3D image; in optimal cases, this can be combined with superresolution microscopy of selected ciliary components as they enter or leave the cilium. This chapter discusses CLEM methods. In the method described in detail, samples are completely processed for sequential fluorescence and SEM observation. This method is ideal for robust antibody localization and minimizes image manipulation in correlating the fluorescent and SEM images. Alternative methods prepare samples for fluorescence imaging followed by processing for SEM then observation in the SEM. This method is ideal for optimal fluorescence imaging, particularly live cell imaging.

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References

  1. Piperno G, Fuller MT (1985) Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms. J Cell Biol 101:2085–2094

    Article  CAS  Google Scholar 

  2. Bishop GA, Berbari NF, Lewis J, Mykytyn K (2007) Type III adenylyl cyclase localizes to primary cilia throughout the adult mouse brain. J Comp Neurol 505:562–571

    Article  Google Scholar 

  3. Caspary T, Larkins CE, Anderson KV (2007) The graded response to sonic Hedgehog depends on cilia architecture. Dev Cell 12:767–778

    Article  CAS  Google Scholar 

  4. Satir P, Pedersen LP, Christensen ST (2010) The primary cilium at a glance. J Cell Sci 123:499–503

    Article  CAS  PubMed Central  Google Scholar 

  5. Lechtreck KF, Gould TJ, Witman GB (2013) Flagellar central pair assembly in Chlamydomonas reinhardtii. Cilia 2:15

    Article  PubMed Central  Google Scholar 

  6. Yang TT, Su J et al (2015) Superresolution pattern recognition reveals the architectural map of the ciliary transition zone. Sci Rep 5:Article 14096

    Article  Google Scholar 

  7. de Boer P, Hoogenboom JP, Giepmans BNG (2015) Correlated light and electron microscopy: ultrastructure lights up! Nat Methods 12:503–513

    Article  CAS  Google Scholar 

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Correspondence to Peter Satir .

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© 2016 Springer Science+Business Media New York

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Macaluso, F.P., Perumal, G.S., Kolstrup, J., Satir, P. (2016). CLEM Methods for Studying Primary Cilia. In: Satir, P., Christensen, S. (eds) Cilia. Methods in Molecular Biology, vol 1454. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3789-9_12

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  • DOI: https://doi.org/10.1007/978-1-4939-3789-9_12

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3787-5

  • Online ISBN: 978-1-4939-3789-9

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