2. To avoid aggregates, all stock solutions of antibodies should be
centrifuged at 16,000 Â g for 5 min. All working solutions can
be filtered on 0.22 μm filters.
3. The purpose of the post-fixation of the immune complexes is
twofold: first, avoiding cleavage of disulfide bounds of the
antibodies (destroying the immunoreactivity) in the mounting
buffers that often contain reducing agents like cysteamine or
β-mercaptoethanol. Moreover, cysteamine solutions are also
weakly basic (pH 8–8.5), weakening the bindings. Second,
reducing the mobility of the antibodies on the antigens (off
and on reactions) that may affect the quality of super-resolution
images.
Acknowledgments
We thank Yves Lutz from the Imaging Centre for discussions on the
adaptation of a Leica protocol and his contribution during earlier
published work. This work was supported by CNRS, Association
pour la Recherche sur le Cancer (ARC), Institut National du Cancer (INCa), Ligue nationale contre le cancer (Ligue), Agence
National pour la Recherche (ANR), and the USIAS research fellowship program of the University of Strasbourg. The superresolution microscope setup was supported by the Alsace Region
and by the French Infrastructure for Integrated Structural Biology
(FRISBI; ANR-10-INSB-05-01) and Instruct-ERIC.
References
1. Orlov I, Myasnikov AG, Andronov L et al
(2017) The integrative role of cryo electron
microscopy in molecular and cellular structural
biology. Biol Cell 109:81–93
2. Rust MJ, Bates M, Zhuang X (2006) Stochastic
optical reconstruction microscopy (STORM)
provides sub-diffraction-limit image resolution. Nat Methods 3:793–795
3. van de Linde S, Lo ¨schberger A, Klein T et al
(2011) Direct stochastic optical reconstruction
microscopy with standard fluorescent probes.
Nat Protoc 6:991–1009
4. Betzig E, Patterson GH, Sougrat R et al (2006)
Imaging intracellular fluorescent proteins at
nanometer
resolution.
Science
313:1642–1645
5. Sharonov A, Hochstrasser RM (2006) Widefield subdiffraction imaging by accumulated
binding of diffusing probes. Proc Natl Acad
Sci U S A 103:18911–18916
6. Dempsey GT, Vaughan JC, Chen KH et al
(2011) Evaluation of fluorophores for optimal
performance in localization-based super-resolution imaging. Nat Methods 8:1027–1036
7. Lippincott-Schwartz J, Patterson GH (2009)
Photoactivatable fluorescent proteins for
diffraction-limited and super-resolution imaging. Trends Cell Biol 19:555–565
8. Owen DM, Rentero C, Rossy J et al (2010)
PALM imaging and cluster analysis of protein
heterogeneity at the cell surface. J Biophotonics 3:446–454
9. Andronov L, Orlov I, Lutz Y et al (2016) ClusterViSu, a method for clustering of protein
complexes by Voronoi tessellation in superresolution microscopy. Sci Rep 6:24084
10. Malkusch S, Endesfelder U, Mondry J et al
(2012) Coordinate-based colocalization analysis of single-molecule localization microscopy
data. Histochem Cell Biol 137:1–10
11. Lee S-H, Shin JY, Lee A et al (2012) Counting
single photoactivatable fluorescent molecules
by photoactivated localization microscopy
284
Leonid Andronov et al.
centrifuged at 16,000 Â g for 5 min. All working solutions can
be filtered on 0.22 μm filters.
3. The purpose of the post-fixation of the immune complexes is
twofold: first, avoiding cleavage of disulfide bounds of the
antibodies (destroying the immunoreactivity) in the mounting
buffers that often contain reducing agents like cysteamine or
β-mercaptoethanol. Moreover, cysteamine solutions are also
weakly basic (pH 8–8.5), weakening the bindings. Second,
reducing the mobility of the antibodies on the antigens (off
and on reactions) that may affect the quality of super-resolution
images.
Acknowledgments
We thank Yves Lutz from the Imaging Centre for discussions on the
adaptation of a Leica protocol and his contribution during earlier
published work. This work was supported by CNRS, Association
pour la Recherche sur le Cancer (ARC), Institut National du Cancer (INCa), Ligue nationale contre le cancer (Ligue), Agence
National pour la Recherche (ANR), and the USIAS research fellowship program of the University of Strasbourg. The superresolution microscope setup was supported by the Alsace Region
and by the French Infrastructure for Integrated Structural Biology
(FRISBI; ANR-10-INSB-05-01) and Instruct-ERIC.
References
1. Orlov I, Myasnikov AG, Andronov L et al
(2017) The integrative role of cryo electron
microscopy in molecular and cellular structural
biology. Biol Cell 109:81–93
2. Rust MJ, Bates M, Zhuang X (2006) Stochastic
optical reconstruction microscopy (STORM)
provides sub-diffraction-limit image resolution. Nat Methods 3:793–795
3. van de Linde S, Lo ¨schberger A, Klein T et al
(2011) Direct stochastic optical reconstruction
microscopy with standard fluorescent probes.
Nat Protoc 6:991–1009
4. Betzig E, Patterson GH, Sougrat R et al (2006)
Imaging intracellular fluorescent proteins at
nanometer
resolution.
Science
313:1642–1645
5. Sharonov A, Hochstrasser RM (2006) Widefield subdiffraction imaging by accumulated
binding of diffusing probes. Proc Natl Acad
Sci U S A 103:18911–18916
6. Dempsey GT, Vaughan JC, Chen KH et al
(2011) Evaluation of fluorophores for optimal
performance in localization-based super-resolution imaging. Nat Methods 8:1027–1036
7. Lippincott-Schwartz J, Patterson GH (2009)
Photoactivatable fluorescent proteins for
diffraction-limited and super-resolution imaging. Trends Cell Biol 19:555–565
8. Owen DM, Rentero C, Rossy J et al (2010)
PALM imaging and cluster analysis of protein
heterogeneity at the cell surface. J Biophotonics 3:446–454
9. Andronov L, Orlov I, Lutz Y et al (2016) ClusterViSu, a method for clustering of protein
complexes by Voronoi tessellation in superresolution microscopy. Sci Rep 6:24084
10. Malkusch S, Endesfelder U, Mondry J et al
(2012) Coordinate-based colocalization analysis of single-molecule localization microscopy
data. Histochem Cell Biol 137:1–10
11. Lee S-H, Shin JY, Lee A et al (2012) Counting
single photoactivatable fluorescent molecules
by photoactivated localization microscopy
284
Leonid Andronov et al.