Abstract
There are an increasing number of experiments to study programmed cell death/apoptosis, one of the characteristics of which is DNA fragmentation. The only current method for in situ detection of DNA fragmentation is Terminal deoxynucleotidyl transferase mediated-dUTP Nick End Labeling, TUNEL. In this study, a new method for in situ detection of apoptotic DNA fragments, namely In Situ Hybridization Chain Reaction, isHCR, was established. The principle of the assay is that the sticky end sequence of the apoptotic cell DNA fragment non-specifically initiates a hybridization chain reaction that specifically detects the apoptotic cell. The results of the combined TUNEL and isHCR method demonstrated that the majority of isHCR-positive cells were also labeled by TUNEL. In situ HCR often detect DNA fragments in the cytoplasm that the classical TUNEL method couldnot, and these cells may be in the early stages of apoptosis. It also indicates that DNA fragments are transferred to the cytoplasm during apoptosis. Because the staining process does not require terminal deoxynucleotidyl transferase as TUNEL staining does, isHCR staining cost low and can be performed on a large number of tissue specimens. It is believed that isHCR has the potential to detect DNA fragmentation of apoptotic cells in situ.
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Funding
This work was supported by the National Natural Science Foundation of the People’s Republic of China (82171220, 81971046 to H Yuan; 818471260 to Z Xiang; 31900647 to R Ji; 81901123 to Y Lu).
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MY main experimental execution; analyzed the data. RJ main experimental execution; analyzed the data. ZZ main experimental execution; making animal models. WW Animal care; animal perfusion; making animal models. YL designed experiments; wrote the manuscript. ZX design the hairpins, wrote the manuscript. HY designed experiments; wrote the manuscript.
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Yang, M., Ji, R., Zhao, Z. et al. Detection of apoptotic cells based on in situ hybridization chain reaction using specific hairpins. Apoptosis 28, 222–232 (2023). https://doi.org/10.1007/s10495-022-01782-5
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DOI: https://doi.org/10.1007/s10495-022-01782-5