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Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)
Published on: September 24, 2015
Electron microscopic in situ DNA nick end-labeling in combination with immunoelectron microscopy
A Ishida-Yamamoto1, T Yamauchi, H Tanaka
1Department of Dermatology, Asahikawa Medical College, Asahikawa, Japan.
Summary
We developed a new electron microscopy method to label DNA nicks in situ. This technique enhances the study of apoptosis and cell differentiation in tissues like skin.
Area of Science:
- Electron Microscopy
- Molecular Biology
- Cell Biology
Background:
- Apoptosis is a crucial biological process.
- Identifying apoptotic cells at the ultrastructural level is challenging.
- Current methods may not preserve cellular architecture.
Purpose of the Study:
- To develop an in situ DNA nick end-labeling method for electron microscopy.
- To combine this method with immunoelectron microscopy.
- To investigate apoptosis in normal skin and Bowen's disease.
Main Methods:
- Cryofixation, freeze-substitution, and Lowicryl K11M embedding of skin tissues.
- In situ labeling of DNA nicks using digoxigenin-dUTP and terminal deoxynucleotidyl transferase.
- Detection of digoxigenin with anti-digoxigenin antibodies conjugated to colloidal gold.
Main Results:
- Specific labeling of DNA nicks in condensed chromatin of differentiated cells and dyskeratotic cells.
- Significantly higher labeling density in apoptotic cells compared to mitotic or cytoplasmic areas.
- Well-preserved ultrastructure and successful double staining with anti-keratin antibody.
Conclusions:
- The developed method allows for electron microscopic visualization of DNA nicks in situ.
- It effectively identifies apoptotic cells and aids in understanding apoptosis mechanisms.
- The technique is versatile and compatible with immunoelectron microscopy.
