Summary
TUNEL-based assays were used to demonstrate the presence of apoptotic cells in tissue sections derived from target tissues of animal models of different diseases. Emphasis was placed on tissue preparation and fixation, as these are crucial to successful histological staining. The protocol suggested here facilitates not only the reliable detection of TUNEL-positive cells but the immunodetection of different proteins in these cells and the surrounding tissues by DAB or fluorescence-based immunostaining.
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References
Taylor, R. C., Cullen, S. P., and Martin, S. J. (2008). Apoptosis: controlled demolition at the cellular level, Nature reviews 9, 231–241.
Willingham, M. C. (1999). Cytochemical methods for the detection of apoptosis, J Histochem Cytochem 47, 1101–1110.
Labat-Moleur, F., Guillermet, C., Lorimier, P., Robert, C., Lantuejoul, S., Brambilla, E., and Negoescu, A. (1998). TUNEL apoptotic cell detection in tissue sections: critical evaluation and improvement, J Histochem Cytochem 46, 327–334.
Fondevila, C., Shen, X. D., Tsuchiyashi, S., Yamashita, K., Csizmadia, E., Lassman, C., Busuttil, R. W., Kupiec-Weglinski, J. W., and Bach, F. H. (2004). Biliverdin therapy protects rat livers from ischemia and reperfusion injury, Hepatology (Baltimore, Md) 40, 1333–1341.
Ito, Y., Shibata, M. A., Kusakabe, K., and Otsuki, Y. (2006). Method of specific detection of apoptosis using formamide-induced DNA denaturation assay, J Histochem Cytochem 54, 683–692.
Allen, R. T., Hunter, W. J., III, and Agrawal, D. K. (1997). Morphological and biochemical characterization and analysis of apoptosis, J Pharmacol Toxicol Meth 37, 215–228.
Barrett, K. L., Willingham, J. M., Garvin, A. J., and Willingham, M. C. (2001). Advances in cytochemical methods for detection of apoptosis, J Histochem Cytochem 49, 821–832.
Gown, A. M., and Willingham, M. C. (2002). Improved detection of apoptotic cells in archival paraffin sections: immunohistochemistry using antibodies to cleaved caspase 3, J Histochem Cytochem 50, 449–454.
Miething, F., Hering, S., Hanschke, B., and Dressler, J. (2006). Effect of fixation to the degradation of nuclear and mitochondrial DNA in different tissues, J Histochem Cytochem 54, 371–374.
Hakem, R., and Mak, T. W. (2001). Animal models of tumor-suppressor genes, Annu Rev Genet 35, 209–241.
Griffin, J. L. (2006). Understanding mouse models of disease through metabolomics, Curr Opin Chem Biol 10, 309–315.
Srinivasan, M., Sedmak, D., and Jewell, S. (2002). Effect of fixatives and tissue processing on the content and integrity of nucleic acids, Am J Pathol 161, 1961–1971.
Wester, K., Asplund, A., Backvall, H., Micke, P., Derveniece, A., Hartmane, I., Malmstrom, P. U., and Ponten, F. (2003). Zinc-based fixative improves preservation of genomic DNA and proteins in histoprocessing of human tissues. Laboratory investigation. J Tech Meth Pathol 83, 889–899.
Beckstead, J. H. (1994). A simple technique for preservation of fixation-sensitive antigens in paraffin-embedded tissues, J Histochem Cytochem 42, 1127–1134.
Taylor, C. R. (2006). Standardization in immunohistochemistry: the role of antigen retrieval in molecular morphology, Biotech Histochem 81, 3–12.
Banz, Y., Hess, O. M., Robson, S. C., Csizmadia, E., Mettler, D., Meier, P., Haeberli, A., Shaw, S., Smith, R. A., and Rieben, R. (2007). Attenuation of myocardial reperfusion injury in pigs by Mirococept, a membrane-targeted complement inhibitor derived from human CR1, Cardiovascular Res 76, 482–493.
Fondevila, C., Shen, X. D., Tsuchiyashi, S., Yamashita, K., Csizmadia, E., Lassman, C., Busuttil, R. W., Kupiec-Weglinski, J. W., and Bach, F. H. (2004). Biliverdin therapy protects rat livers from ischemia and reperfusion injury, Hepatology (Baltimore, Md) 40, 1333–1341.
Patel, V. I., Daniel, S., Longo, C. R., Shrikhande, G. V., Scali, S. T., Czismadia, E., Groft, C. M., Shukri, T., Motley-Dore, C., Ramsey, H. E., Fisher, M. D., Grey, S. T., Arvelo, M. B., and Ferran, C. (2006). A20, a modulator of smooth muscle cell proliferation and apoptosis, prevents and induces regression of neointimal hyperplasia, Faseb J 20, 1418–1430.
Onishi, S., Miyata, H., Inamoto, T., Qi, W. M., Yamamoto, K., Yokoyama, T., Warita, K., Hoshi, N., and Kitagawa, H. (2007). Immunohistochemical study on the delayed progression of epithelial apoptosis in follicle-associated epithelium of rat Peyer’s patch, J Vet Med Sci 69, 1123–1129.
Bartels-Stringer, M., Kramers, C., Wetzels, J. F., Russel, F. G., Groot, H., and Rauen, U. (2003). Hypothermia causes a marked injury to rat proximal tubular cells that is aggravated by all currently used preservation solutions, Cryobiology 47, 82–91.
Soares, M. P., Lin, Y., Anrather, J., Csizmadia, E., Takigami, K., Sato, K., Grey, S. T., Colvin, R. B., Choi, A. M., Poss, K. D., and Bach, F. H. (1998). Expression of heme oxygenase-1 can determine cardiac xenograft survival, Nat Med 4, 1073–1077.
Evans, P. C., Taylor, E. R., and Kilshaw, P. J. (2001). Signaling through CD31 protects endothelial cells from apoptosis, Transplantation 71, 457–460.
Acknowledgments
The authors thank Christiane Ferran, Leo Otterbein, and Beek Chin at Beth Israel Deaconess Hospital of Harvard Medical School for their continuous support of the work presented in this publication. The work was financially supported by RO1 grants HL08013, DK063275. We thank also Alexis Khalil for critical reading of the manuscript.
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Csizmadia, E., Csizmadia, V. (2009). Detection of Apoptosis in Tissue Sections. In: Erhardt, P., Toth, A. (eds) Apoptosis. Methods in Molecular Biology, vol 559. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-017-5_4
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DOI: https://doi.org/10.1007/978-1-60327-017-5_4
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