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Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging
Published on: April 28, 2021
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A simple histological technique to improve immunostaining when using DNA denaturation for BrdU labelling
Jenna J Boulanger1, William A Staines2, Véronique LeBlanc1
1School of Psychology, University of Ottawa, 136 Jean-Jacques Lussier, Ottawa, ON, Canada K1 N 6N5.
Journal of Neuroscience Methods
|December 2, 2015
Summary
A new post-fixation step protects tissue antigens during DNA denaturation for 5-bromo-2'-deoxyuridine (BrdU) staining. This method improves immunohistochemistry, preventing false negatives and enabling detection of more proteins in cell proliferation studies.
Area of Science:
- Neuroscience
- Cell Biology
- Immunohistochemistry
Background:
- Standard immunohistochemistry for 5-bromo-2 -deoxyuridine (BrdU) requires DNA denaturation, which can damage tissue and reduce antigenicity.
- This damage leads to weak immunostaining and potential false negatives, limiting the detection of specific proteins alongside BrdU.
- Investigating oligodendrocyte progenitor cell differentiation requires reliable detection of multiple markers.
Purpose of the Study:
- To develop a method that protects antigens during DNA denaturation for combined BrdU and other protein staining.
- To improve the reliability of immunohistochemistry for studying cell proliferation and differentiation.
- To enable simultaneous detection of BrdU incorporation and specific cellular markers in complex tissues.
Main Methods:
- An overnight post-fixation step was incorporated after primary antibody incubation and before acid/heat DNA denaturation.
- Immunohistochemistry was performed on NG2CreER:EYFP reporter mice to study oligodendrocyte progenitor cell differentiation.
- Double immunostaining for EYFP (using anti-GFP antibody), Olig1, and GST-pi was conducted with and without the post-fixation step.
Main Results:
- DNA denaturation significantly reduced immunostaining for GFP, Olig1, and GST-pi.
- The overnight post-fixation step effectively protected these antigens from damage caused by DNA denaturation.
- This protection prevented the observed fading and false negatives, preserving signal integrity for multiple markers.
Conclusions:
- Incorporating an overnight post-fixation step is a simple yet effective method to preserve antigenicity during BrdU staining protocols.
- This technique enhances the reliability of immunohistochemistry, allowing for the successful detection of a broader range of proteins.
- The improved method facilitates more comprehensive studies of cell proliferation and differentiation, particularly in complex biological systems.
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