PFA fixation enables artifact-free super-resolution imaging of the actin cytoskeleton and associated proteins

Super-resolution microscopy (SRM) allows precise localization of proteins in cellular organelles and structures, including the actin cytoskeleton. Yet sample preparation protocols for SRM are rather anecdotal and still being optimized. Thus, SRM-based imaging of the actin cytoskeleton and associated...

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Bibliographic Details
Published in:Biology open Vol. 5; no. 7; pp. 1001 - 1009
Main Authors: Leyton-Puig, Daniela, Kedziora, Katarzyna M., Isogai, Tadamoto, van den Broek, Bram, Jalink, Kees, Innocenti, Metello
Format: Journal Article
Language:English
Published: England The Company of Biologists Ltd 15.07.2016
The Company of Biologists
Subjects:
Actin
Actin cytoskeleton
Cellular structure
Clathrin
Cytoskeleton
dSTORM
Fixation
Image resolution
Lamellipodia
Localization
Methods & Techniques
Organelles
Protein localization
Proteins
Pseudopodia
Regulatory proteins
Reproducibility
Sample preparation
Super-resolution microscopy (SRM)
Protein localization
dSTORM
Fixation
Actin cytoskeleton
Super-resolution microscopy (SRM)
ISSN:2046-6390, 2046-6390
Online Access:Get full text
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Summary:Super-resolution microscopy (SRM) allows precise localization of proteins in cellular organelles and structures, including the actin cytoskeleton. Yet sample preparation protocols for SRM are rather anecdotal and still being optimized. Thus, SRM-based imaging of the actin cytoskeleton and associated proteins often remains challenging and poorly reproducible. Here, we show that proper paraformaldehyde (PFA)-based sample preparation preserves the architecture of the actin cytoskeleton almost as faithfully as gold-standard glutaraldehyde fixation. We show that this fixation is essential for proper immuno-based localization of actin-binding and actin-regulatory proteins involved in the formation of lamellipodia and ruffles, such as mDia1, WAVE2 and clathrin heavy chain, and provide detailed guidelines for the execution of our method. In summary, proper PFA-based sample preparation increases the multi-color possibilities and the reproducibility of SRM of the actin cytoskeleton and its associated proteins.
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These authors contributed equally to this work
ISSN:2046-6390
2046-6390
DOI:10.1242/bio.019570