Combination of TUNEL Assay with Immunohistochemistry for Simultaneous Detection of DNA Fragmentation and Oxidative Cell Damage

  • Alexander E. Kalyuzhny
Part of the Methods in Molecular Biology book series (MIMB, volume 682)


Oxidative cell damage causes disruption of DNA via formation of 8-hydroxy-2′-deoxyguanosine and can trigger apoptotic cell death. The cells damaged by oxidative stress can either become apoptotic, or recover. Therefore, it is helpful to employ a parallel assay that would confirm whether cells experiencing oxidative damage undergo apoptosis. Our paper describes the technique that combines immunohistochemical detection of 8-hydroxy-2′-deoxyguanosine with the TUNEL assay. This permits simultaneous detection of oxidative damage and apoptosis at a single-cell level. We have developed simple and reliable protocols which can be used with cultured cells and slide-mounted tissue sections. These techniques can be employed in research dealing with high-throughput drug screening, toxicology, and cancer.

Key words

Oxidative stress, 8-hydroxy-2′-deoxyguanosine Immunohistochemistry TUNEL assay Apoptosis Cultured cells Tissue sections Double-labeling 


  1. 1.
    Muppidi, J., Porter, M., and Siegel, R. M. (2004) Measurement of apoptosis and other forms of cell death. Curr. Protoc. Immunol. Chapter, Unit 3.17.Google Scholar
  2. 2.
    Kerr, W. G. (2008) Analysis of apoptosis in hematopoietic stem cells by flow cytometry. Methods Mol. Biol. 430, 87–99.PubMedCrossRefGoogle Scholar
  3. 3.
    Alarcon, V. B., and Marikawa, Y. (2004) Molecular study of mouse peri-implantation development using the in vitro culture of aggregated inner cell mass. Mol. Reprod. Dev. 67, 83–90.PubMedCrossRefGoogle Scholar
  4. 4.
    Goncalves, J. S., Sasso-Cerri, E., and Cerri, P. S. (2008) Cell death and quantitative reduction of rests of Malassez according to age. J. Periodontal Res. 43, 478–481.PubMedCrossRefGoogle Scholar
  5. 5.
    Harada, K., Ferdous, T., Itashiki, Y., Takii, M., Mano, T., Mori, Y., and Ueyama, Y. (2009) Effects of cepharanthine alone and in combination with fluoropyrimidine anticancer agent, S-1, on tumor growth of human oral squamous cell carcinoma xenografts in nude mice. Anticancer Res. 29, 1263–1270.PubMedGoogle Scholar
  6. 6.
    Kang, Y., Zhang, X., Jiang, W., Wu, C., Chen, C., Zheng, Y., Gu, J., and Xu, C. (2009) Tumor-directed gene therapy in mice using a composite nonviral gene delivery system consisting of the piggyBac transposon and polyethylenimine. BMC Cancer 9, 126.PubMedCrossRefGoogle Scholar
  7. 7.
    Anderson, A. J., Stoltzner, S., Lai, F., Su, J., and Nixon, R. A. (2000) Morphological and biochemical assessment of DNA damage and apoptosis in Down syndrome and Alzheimer disease, and effect of postmortem tissue archival on TUNEL. Neurobiol. Aging 21, 511–524.PubMedCrossRefGoogle Scholar
  8. 8.
    Lee, S. Y., Moon, Y., Hee Choi, D., Jin Choi, H., and Hwang, O. (2007) Particular vulnerability of rat mesencephalic dopaminergic neurons to tetrahydrobiopterin: Relevance to Parkinson’s disease. Neurobiol. Dis. 25, 112–120.PubMedCrossRefGoogle Scholar
  9. 9.
    Kadota, T., Shingo, T., Yasuhara, T., Tajiri, N., Kondo, A., Morimoto, T., Yuan, W. J., Wang, F., Baba, T., Tokunaga, K., Miyoshi, Y., and Date, I. (2009) Continuous intraventricular infusion of erythropoietin exerts neuroprotective/rescue effects upon Parkinson’s disease model of rats with enhanced neurogenesis. Brain Res. 1254, 120–127.PubMedCrossRefGoogle Scholar
  10. 10.
    Ohta, Y., Kamiya, T., Nagai, M., Nagata, T., Morimoto, N., Miyazaki, K., Murakami, T., Kurata, T., Takehisa, Y., Ikeda, Y., Asoh, S., Ohta, S., and Abe, K. (2008) Therapeutic benefits of intrathecal protein therapy in a mouse model of amyotrophic lateral sclerosis. J. Neurosci. Res. 86, 3028–3037.PubMedCrossRefGoogle Scholar
  11. 11.
    Park, J. E., Lee, S. T., Im, W. S., Chu, K., and Kim, M. (2008) Galantamine reduces striatal degeneration in 3-nitropropionic acid model of Huntington’s disease. Neurosci. Lett. 448, 143–147.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Alexander E. Kalyuzhny
    • 1
  1. 1.R&D Systems, IncMinneapolisUSA

Personalised recommendations