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Method Article
Tips and tricks for artifact-free PFA-based fixation of the actin cytoskeleton and its regulatory proteins for single molecule localization super-resolution microscopy
Metello Innocenti
Jalink / Innocenti Lab (Netherlands Cancer Institute)
Corresponding Author
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Super-resolution microscopy (SRM) is a vital tool for the analysis of the architecture of actin cytoskeleton and detailed mapping localization of actin-binding proteins in cells. However, optimal sample preparation and imaging conditions for SRM have remained rather anecdotal, making SRM-based imaging of the actin cytoskeleton and associated proteins technically challenging and poorly reproducible.
Here we describe a protocol for high-resolution SRM imaging of the actin cytoskeleton and actin-binding proteins that preserves the architecture of actin-based protrusions, using as a fixative paraformaldehyde. This procedure is compatible with the analysis of both endogenous and genetically encoded fluorescent proteins thereby expanding the options for antibody labeling.
This straightforward protocol allows to obtain artifact-free super-resolution images of the actin cytoskeleton and actin-binding proteins in only three days.
Keywords
super-resolution, dSTORM, GSDIM, actin, fixation, sample preparation
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Table 1 Trobleshooting Common problems and solution thereof.
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Associated Publications
Initiation of lamellipodia and ruffles involves cooperation between mDia1 and the Arp2/3 complex
by T. Isogai, R. van der Kammen, D. Leyton-Puig, +3
Journal Of Cell Science (21 March, 2016)
PFA fixation enables artifact-free super-resolution imaging of the actin cytoskeleton and associated proteins
by Daniela Leyton-Puig, Katarzyna M. Kedziora, Tadamoto Isogai, +3
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This is a preprint. It has not completed peer review.
Method Article
Tips and tricks for artifact-free PFA-based fixation of the actin cytoskeleton and its regulatory proteins for single molecule localization super-resolution microscopy
Metello Innocenti
Jalink / Innocenti Lab (Netherlands Cancer Institute)
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 14 Jul, 2016
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
Super-resolution microscopy (SRM) is a vital tool for the analysis of the architecture of actin cytoskeleton and detailed mapping localization of actin-binding proteins in cells. However, optimal sample preparation and imaging conditions for SRM have remained rather anecdotal, making SRM-based imaging of the actin cytoskeleton and associated proteins technically challenging and poorly reproducible.
Here we describe a protocol for high-resolution SRM imaging of the actin cytoskeleton and actin-binding proteins that preserves the architecture of actin-based protrusions, using as a fixative paraformaldehyde. This procedure is compatible with the analysis of both endogenous and genetically encoded fluorescent proteins thereby expanding the options for antibody labeling.
This straightforward protocol allows to obtain artifact-free super-resolution images of the actin cytoskeleton and actin-binding proteins in only three days.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Associated Publications
Initiation of lamellipodia and ruffles involves cooperation between mDia1 and the Arp2/3 complex
by T. Isogai, R. van der Kammen, D. Leyton-Puig, +3
Journal Of Cell Science (21 March, 2016)
PFA fixation enables artifact-free super-resolution imaging of the actin cytoskeleton and associated proteins
by Daniela Leyton-Puig, Katarzyna M. Kedziora, Tadamoto Isogai, +3