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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.
Insights
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.
Abstract:
We describe an in situ DNA nick end-labeling method that can be performed at the electron microscopic level and can also be combined with immunoelectron microscopy. As the materials, we used skin tissues from normal skin and from Bowen's disease that had been cryofixed, freeze-substituted, and embedded in Lowicryl K11M resin. Ultrathin sections were cut and incubated with a reaction buffer containing digoxigenin-dUTP and terminal deoxynucleotidyl transferase. Digoxigenin nucleotides were labeled with anti-digoxigenin antibodies conjugated with colloidal gold. Specific signals were detected in the condensed chromatin of differentiated epidermal cells and hair follicles in normal skin and of dyskeratotic cells in Bowen's disease. The labeling density over chromosomal areas of apoptotic cells was significantly higher than that over chromosomal areas of mitotic cells or cytoplasmic areas. Ultrastructure was well preserved and double staining with an anti-keratin antibody was also successfully performed. This simple method has a wide range of applications to identify the nature of apoptotic cells and explore the mechanisms of apoptosis.
