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Simultaneous in situ hybridization and TUNEL to identify cells undergoing apoptosis
B R Gochuico1, M C Williams, A Fine
1Department of Biochemistry and Anatomy, Boston University School of Medicine, MA 02118, USA.
Abstract:
Apoptotic cells in tissue sections can be localized by in situ labelling of partly degraded DNA. In a heterogeneous population of cells, however, the specific identity of cell types undergoing apoptosis often cannot be reliably achieved at the light microscope level because of the marked alterations in cellular morphology that characterize apoptosis. In order to clearly specify cell types undergoing apoptosis, in situ end labelling has been coupled to immunohistochemistry. This method is limited by the availability of antibodies that bind to cell-specific protein markers in tissue sections. In contrast, we describe a method that combines in situ end labellin with in situ hybridization, a technique that specifies cell types based on mRNA expression. Taking advantage of the specific expression of surfactant protein C mRNA in type II alveolar epithelial cells, we demonstrate that this technique has the ability to localize alveolar type II cells undergoing apoptosis in vivo after the intratracheal instillation of an antibody that activates the cell surface Fas protein. The wide availability of cell-specific gene markers suggests that this method can be adapted to define cell types that undergo apoptosis during various physiological and pathological states in vivo.
Insights
This study introduces a new method combining in situ end labeling with in situ hybridization to identify specific cell types undergoing apoptosis. This technique accurately localizes apoptotic alveolar type II cells using surfactant protein C mRNA markers.
Area of Science:
- Cell Biology
- Molecular Biology
- Pathology
Background:
- Identifying specific cell types undergoing apoptosis in heterogeneous tissues is challenging due to morphological changes.
- Current methods like immunohistochemistry are limited by antibody availability for cell-specific markers.
Purpose of the Study:
- To develop and validate a novel method for precisely identifying apoptotic cell populations within tissues.
- To overcome the limitations of existing techniques by combining in situ end labeling with in situ hybridization.
Main Methods:
- The study combined in situ end labeling (ISEL) with in situ hybridization (ISH).
- Surfactant protein C mRNA was used as a specific marker for type II alveolar epithelial cells.
- Apoptosis was induced in vivo via intratracheal instillation of an antibody targeting the Fas protein.
Main Results:
- The combined ISEL-ISH method successfully localized alveolar type II cells undergoing apoptosis.
- This technique accurately identified apoptotic type II cells based on surfactant protein C mRNA expression.
- Demonstrated the ability to specify apoptotic cell types in vivo.
Conclusions:
- The novel ISEL-ISH technique enables precise identification of apoptotic cells based on mRNA expression.
- This method overcomes limitations of morphological identification and antibody-dependent techniques.
- The approach is adaptable for studying apoptosis in various cell types during different physiological and pathological conditions.