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Detection of fragmented DNA in apoptotic cells embedded in LR white: A combined histochemical (LM) and
1Department of Anatomy and Cell Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799, USA.
Abstract:
We developed an improved method for the detection of double-strand DNA breaks in apoptotic cells at both the light (LM) and electron microscopic (EM) levels using a modification of the TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick end-labeling (TUNEL) technique. Cultured rat cerebellar granule cells were exposed to low potassium conditions to induce apoptosis. Twenty-four hr after treatment, one group of cells was fixed in situ with 4% paraformaldehyde and labeled for DNA fragmentation characteristic of apoptosis. Apoptotic cells were visualized with diaminobenzidine (DAB) and viewed by LM. The second group of cells was detached from the culture dish, pelleted, fixed with a 4% paraformaldehyde and 0. 2% glutaraldehyde mixture, and embedded in LR White. For LM, the modified TUNEL technique was performed on 1.5-microm LR White sections and apoptotic cells were visualized using an enzymatic reaction to generate a blue precipitate. For EM, thin sections (94 nm) were processed and DNA fragmentation was identified using modified TUNEL with streptavidin-conjugated gold in conjunction with in-depth ultrastructural detail. Alternate sections of cells embedded in LR White can therefore be used for LM and EM TUNEL-based detection of apoptosis. The present findings suggest that the modified TUNEL technique on LR White semithin and consecutive thin sections has useful application for studying the fundamental mechanism of cell death. (J Histochem Cytochem 47:561-568, 1999)
Insights
This study introduces an enhanced TUNEL technique for detecting DNA breaks in apoptotic cells. The modified method allows for improved visualization using both light and electron microscopy, aiding cell death mechanism research.
Area of Science:
- Cell Biology
- Histochemistry
- Neuroscience
Background:
- Apoptosis involves DNA fragmentation, a key indicator of programmed cell death.
- Detecting DNA breaks in apoptotic cells is crucial for understanding cell death mechanisms.
- Existing methods for DNA break detection have limitations in resolution and application.
Purpose of the Study:
- To develop an improved method for detecting double-strand DNA breaks in apoptotic cells.
- To enable detection at both light microscopy (LM) and electron microscopy (EM) levels.
- To facilitate the study of fundamental cell death mechanisms.
Main Methods:
- Modified terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) technique.
- Induction of apoptosis in cultured rat cerebellar granule cells using low potassium conditions.
- Dual application for LM (diaminobenzidine visualization) and EM (streptavidin-conjugated gold labeling) on LR White embedded sections.
Main Results:
- Successful detection of DNA fragmentation characteristic of apoptosis in cultured cells.
- Visualization of apoptotic cells using both LM and EM with the modified TUNEL technique.
- Demonstration that alternate sections can be used for both LM and EM analysis.
Conclusions:
- The modified TUNEL technique offers a versatile approach for studying apoptosis.
- This method enhances the ability to investigate the fundamental mechanisms of cell death.
- The technique is applicable to both semithin and thin sections for comprehensive analysis.