Related Experiment Video
Updated: Nov 9, 2025
Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes
Published on: October 31, 2016
An optimized fixation method containing glyoxal and paraformaldehyde for imaging nuclear bodies
Run-Wen Yao1, Peng-Fei Luan1, Ling-Ling Chen1,2,3
1State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
This study introduces glyoxal (GO) and paraformaldehyde (PFA) fixation, improving RNA and protein imaging in nuclear bodies. This method enhances signal quality and preserves ultrastructure for advanced microscopy.
Area of Science:
- Cell Biology
- Molecular Imaging
- Biochemistry
Background:
- Mammalian cell nuclei contain membrane-less nuclear bodies (NBs) composed of proteins and RNAs.
- Microscopy is crucial for studying NB organization, but current fixation methods compromise RNA or protein signal quality and ultrastructure.
- Existing fixation techniques present a trade-off between RNA fluorescence in situ hybridization (FISH) signal and protein labeling efficiency or structural integrity.
Purpose of the Study:
- To develop an improved fixation method for simultaneous high-quality imaging of RNA and proteins within nuclear bodies.
- To evaluate the efficacy of glyoxal (GO) combined with paraformaldehyde (PFA) fixation for preserving NB ultrastructure and molecular signals.
- To enable multicolor imaging of different nuclear bodies with minimal artifact.
Main Methods:
- Addition of glyoxal (GO) to the standard paraformaldehyde (PFA) fixation protocol.
- Assessment of RNA fluorescence in situ hybridization (FISH) signal intensity and quality.
- Evaluation of protein fluorescent signal preservation during fixation and immunostaining.
- Super-resolution microscopy to analyze the ultrastructure of nuclear bodies post-fixation.
Main Results:
- GO/PFA fixation significantly enhances RNA FISH signals by increasing nuclear permeability and probe accessibility.
- The GO/PFA method largely preserves protein fluorescent signals, overcoming limitations of traditional fixation.
- GO/PFA fixation allows for covisualization of diverse nuclear bodies with minimal impact on their ultrastructures.
- Super-resolution microscopy confirmed the preservation of NB ultrastructure using the novel fixation method.
Conclusions:
- Glyoxal/PFA fixation offers a superior method for preserving both RNA and protein signals in nuclear bodies.
- This technique improves imaging quality and enables detailed ultrastructural analysis of NBs using super-resolution microscopy.
- The developed GO/PFA fixation protocol is valuable for advancing the study of nuclear body organization and function.
