Tips and tricks for artifact-free PFA-based fixation of the actin cytoskeleton and its regulatory proteins for single molecule localization super-resolution microscopy
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.
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Table 1 Trobleshooting Common problems and solution thereof.
Posted 14 Jul, 2016
Tips and tricks for artifact-free PFA-based fixation of the actin cytoskeleton and its regulatory proteins for single molecule localization super-resolution microscopy
Posted 14 Jul, 2016
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
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