Abstract
Higher-order chromatin organization shaped by epigenetic modifications influence the chromatin environment and subsequently regulate gene expression. Direct visualization of the higher-order chromatin structure at their epigenomic states is of great importance for understanding chromatin compaction and its subsequent effect on gene expression and various cellular processes. With the recent advances in super-resolution microscopy, the higher-order chromatin structure can now be directly visualized in situ down to the scale of ~30 nm. This protocol provides detailed description of super-resolution imaging of higher-order chromatin structure using stochastic optical reconstruction microscopy (STORM). We discussed fluorescence staining methods of DNA and histone proteins and crucial technical factors to obtain high-quality super-resolution images.
Keywords: Higher-order chromatin, Epigenetic, Histone modification, Super-resolution, STORM
Background
Recent advances in super-resolution imaging technique provide new potentials to observe the biological structures at the molecular scale. In particular, single-molecule localization based super-resolution technique such as stochastic optical reconstruction microscopy (STORM) has become a valuable tool to directly observe higher-order chromatin structure in situ down to ~20-30 nm resolution (Xu et al., 2018). Although many detailed protocols have been devoted for STORM imaging in general, few have been focused on imaging higher-order chromatin structures. Chromatin has densely packed structure, and the nucleus of mammalian cells tends to be thicker than other membrane-based proteins. Such samples tend to give higher background and present more overlapping fluorescent emitters. Therefore, the proper optimization of nuclear staining for STORM imaging is critical to obtain high-quality super-resolution images of chromatin structure. A compromise in any of staining steps may lead to significant image artifacts and degradation in image resolution. Here we provide detailed protocols for STORM-based super-resolution imaging of higher-order chromatin structure marked by either DNA and histone proteins. We also provide detailed procedures on the conjugation of the fluorophores with secondary antibodies, for single-color and two-color STORM imaging. The protocol presented here is optimized for STORM imaging, we believe that this protocol can also be extended to most high-resolution fluorescence imaging of chromatin structures or other proteins of interest.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Recipes
Acknowledgments
We acknowledge the funding support from National Institute of Health Grant Number R01CA185363 and R33CA225494. Our protocols were adapted based on the previously published protocols (Bates et al., 2007 and 2013; van de Linde et al., 2011).
Competing interests
The authors declare no competing interests.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.