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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| Abstract | 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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| AbstractList | 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.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. 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. Summary: We show that proper PFA fixation allows high-quality super-resolution imaging of the actin cytoskeleton and can outperform gold-standard glutaraldehyde fixation for imaging of actin-binding 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 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. |
| Author | Innocenti, Metello Kedziora, Katarzyna M. Jalink, Kees van den Broek, Bram Isogai, Tadamoto Leyton-Puig, Daniela |
| AuthorAffiliation | 1 Division of Cell Biology I , The Netherlands Cancer Institute , Plesmanlaan 121, Amsterdam 1066 CX , The Netherlands 2 Division of Molecular Genetics , The Netherlands Cancer Institute , Plesmanlaan 121, Amsterdam 1066 CX , The Netherlands |
| AuthorAffiliation_xml | – name: 2 Division of Molecular Genetics , The Netherlands Cancer Institute , Plesmanlaan 121, Amsterdam 1066 CX , The Netherlands – name: 1 Division of Cell Biology I , The Netherlands Cancer Institute , Plesmanlaan 121, Amsterdam 1066 CX , The Netherlands |
| Author_xml | – sequence: 1 givenname: Daniela surname: Leyton-Puig fullname: Leyton-Puig, Daniela organization: Division of Cell Biology I, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1066 CX, The Netherlands – sequence: 2 givenname: Katarzyna M. surname: Kedziora fullname: Kedziora, Katarzyna M. organization: Division of Cell Biology I, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1066 CX, The Netherlands – sequence: 3 givenname: Tadamoto surname: Isogai fullname: Isogai, Tadamoto organization: Division of Molecular Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1066 CX, The Netherlands – sequence: 4 givenname: Bram surname: van den Broek fullname: van den Broek, Bram organization: Division of Cell Biology I, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1066 CX, The Netherlands – sequence: 5 givenname: Kees surname: Jalink fullname: Jalink, Kees organization: Division of Cell Biology I, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1066 CX, The Netherlands – sequence: 6 givenname: Metello orcidid: 0000-0002-7455-2559 surname: Innocenti fullname: Innocenti, Metello organization: Division of Molecular Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1066 CX, The Netherlands |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/27378434$$D View this record in MEDLINE/PubMed |
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| Keywords | Protein localization dSTORM Fixation Actin cytoskeleton Super-resolution microscopy (SRM) |
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| SubjectTerms | 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) |
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| Title | PFA fixation enables artifact-free super-resolution imaging of the actin cytoskeleton and associated proteins |
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