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Visualization of Endogenous Transcription Factors in Single Cells Using an Antibody Electroporation-Based Imaging Approach

  • Sascha ConicEmail author
  • Dominique Desplancq
  • Alexia Ferrand
  • Nacho Molina
  • Etienne Weiss
  • László ToraEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2038)

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.

Key words

Antibodies Antibody delivery Single cells Endogenous proteins RNA polymerase II Transcription Imaging Nucleus Factor distribution 3D structured illumination microscopy (3D-SIM) 

Notes

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Sascha Conic
    • 1
    • 2
    • 3
    • 4
    Email author
  • Dominique Desplancq
    • 4
    • 5
  • Alexia Ferrand
    • 6
  • Nacho Molina
    • 1
    • 2
    • 3
    • 4
  • Etienne Weiss
    • 4
    • 5
  • László Tora
    • 1
    • 2
    • 3
    • 4
    Email author
  1. 1.Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC)IllkirchFrance
  2. 2.Centre National de la Recherche Scientifique, UMR7104IllkirchFrance
  3. 3.Institut National de la Santé et de la Recherche Médicale, U1258IllkirchFrance
  4. 4.Université de StrasbourgIllkirchFrance
  5. 5.Biotechnology and Cell Signaling, UMR 7242IllkirchFrance
  6. 6.Imaging Core Facility, BiozentrumUniversity of BaselBaselSwitzerland

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