Abstract
In this chapter, we describe an antibody electroporation-based imaging approach that allows for precise imaging and quantification of endogenous transcription factor (i.e., RNA Polymerase II) distributions in single cells using 3D structured illumination microscopy (3D-SIM). The labeling is achieved by the efficient and harmless delivery of fluorescent dye-conjugated antibodies into living cells and the specific binding of these antibodies to the targeted factors. Our step-by-step protocol describes the procedure of the labeling of the specific antibodies, their electroporation into living cells, the sample preparation and 3D-SIM imaging as well as the postimaging analyses of the labeled endogenous transcription factors to obtain information about their nuclear distribution as well as their function. This protocol can be applied to a plethora of endogenous nuclear factors by using target specific noninhibiting antibodies.
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Acknowledgments
This work was supported by funds from CNRS, INSERM, University of Strasbourg, Ligue Régionale contre le Cancer (CCIRGE-BFC) (to EW), by the European Research Council (ERC) Advanced grant (ERC-2013-340551, Birtoaction) (to LT) and a grant ANR-10-LABX-0030-INRT, a French State fund managed by the Agence Nationale de la Recherche under the frame program Investissements d’Avenir ANR-10-IDEX-0002-02 (to IGBMC).
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Conic, S., Desplancq, D., Ferrand, A., Molina, N., Weiss, E., Tora, L. (2019). Visualization of Endogenous Transcription Factors in Single Cells Using an Antibody Electroporation-Based Imaging Approach. In: Shav-Tal, Y. (eds) Imaging Gene Expression. Methods in Molecular Biology, vol 2038. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9674-2_14
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DOI: https://doi.org/10.1007/978-1-4939-9674-2_14
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