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Published on: September 3, 2017
A restaining method to restore faded fluorescence in tissue specimens for quantitative confocal microscopy
André Huisman1, Lennert S Ploeger, Hub F J Dullens
1Department of Pathology, University Medical Center, Utrecht, The Netherlands.
Summary
This study developed a protocol to remove and restain TO-PRO-3 fluorescent dye on tissue sections. This method allows repeated DNA content analysis using confocal laser scanning microscopy (CLSM) on valuable samples.
Area of Science:
- Biomedical Imaging
- Histology
- Molecular Biology
Background:
- Confocal laser scanning microscopy (CLSM) is crucial for analyzing DNA content and distribution in tissue sections.
- Repeated measurements on valuable clinical samples are often necessary but challenging due to staining permanence.
Purpose of the Study:
- To develop a reliable protocol for removing and restaining TO-PRO-3 fluorescent dye on tissue sections.
- To enable repeated DNA content and distribution analysis on the same tissue sections using CLSM.
Main Methods:
- Paraffin-embedded adrenal tissue sections were stained with TO-PRO-3 and imaged via CLSM.
- Three destaining methods involving incubation at varying temperatures and durations were tested.
- Sections were re-imaged to assess residual fluorescence and then restained and re-imaged for comparison.
Main Results:
- Incubation at 37°C in preheated medium twice for 20 minutes yielded optimal destaining.
- Post-destaining and restaining, 3-D texture features of nuclei remained comparable to initial staining.
- Minimal influence on fluorescence intensity and nuclear chromatin distribution was observed.
Conclusions:
- A validated protocol exists for effective TO-PRO-3 removal and restaining in tissue sections.
- This technique facilitates repeated quantitative analysis of DNA content and chromatin structure.
- The method preserves tissue integrity, crucial for analyzing precious clinical samples.
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