Specific Methods for Detection and Quantification of Apoptosis in Tissue Sections

  • Matthew A. Wallig
  • Curtis M. Chan
  • Nancy A. Gillett
Part of the Methods in Pharmacology and Toxicology book series (MIPT)


Apoptosis as a distinct pathologic process has been recognized for decades. Its importance in many disease processes has become increasingly appreciated as new techniques for detecting and quantifying it have been developed with almost exponential rapidity. While all these techniques have their advantages and disadvantages, it is surprising how often simple morphologic assessment is overlooked in the rush to develop ever more sophisticated and “glitzy” techniques. With the appropriate training, apoptosis can be evaluated and even semi-quantified by simply examining a standard hematoxylin-and eosin-stained section closely and carefully. Admittedly, apoptosis (also termed apoptotic necrosis) can be harder to detect morphologically than necrosis (also termed oncotic necrosis). Its rapid progression once triggered (usually minutes), the rapid disposition of the apoptotic cells via ingestion by adjacent cells or resident macrophages (often just several hours), and the participation of only small numbers of cells at any one time during the process can make detecting apoptosis challenging. However, there are unique morphologic features associated with the process that an experienced morphologist can easily and rapidly detect to obtain a “global,” if not truly quantitative assessment of the degree of apoptosis occurring in a particular tissue. Simple morphologic assessment offers the advantages of giving the investigator an idea of the distribution of apoptosis within a tissue in the context of “real life” as well as the specific cell types involved within that tissue.


Apoptotic Cell Apoptotic Body Initiator Caspases Ultrastructural Examination Nuclear Profile 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Renvoize, C., Biola, A., Pallardy, M., and Breard, J. (1998) Apoptosis: identification of dying cells. Cell Biol. Toxicol. 14 (2), 111–120.PubMedCrossRefGoogle Scholar
  2. 2.
    Wyllie, A. H., Kerr, J. F. R., and Currie, A. R. (1980) Cell death: the significance of apoptosis. Int. Rev. Cytol. 68, 251–307.PubMedCrossRefGoogle Scholar
  3. 3.
    Bursch, W., Tuper, H. S., Lauer, B., and Schulte-Harmann, R. (1985) Quantitative histological and histochemical studies on the occurrence and stages of controlled cell death (apoptosis) during regression of rat liver hyperlasia. Virchows Arch. [Cell Pathol.] 50, 153–166.CrossRefGoogle Scholar
  4. 4.
    Goldsworthy, T. L., Franssen-Steen, R., and Maronpot, R. R. (1996) Importance of and approaches to quantification of hepatocyte apoptosis. Toxicol. Pathol. 24(1), 24–35.PubMedCrossRefGoogle Scholar
  5. 5.
    Majno, G., and Joris, I. (1995) Apoptosis, oncosis and necrosis: an overview of cell death. Amer. J. Pathol. 146, 3–15.Google Scholar
  6. 6.
    Huppertz, B, Frank, HG, Kauffmann, P. (1999) The apoptosis cascade: morphological and immunohistochemical methods for its visualization. Anat. Embryol. 200, 1–18.PubMedCrossRefGoogle Scholar
  7. 7.
    Velier, J., Ellison, J., Kikly, K., Spera, P., Barone, F., Feuerstein, G. (1999) Caspase-8 and Caspase-3 are expressed by different populations of cortical neurons undergoing delayed cell death after focal stroke in the rat. J. Neurosci. 19(14), 5932–5941.PubMedGoogle Scholar
  8. 8.
    Soini, Y., and Paakko, P. (1999) Apoptosis and expression of caspases 3,6 and 8 in laignant non-Hodgkin’s lymphomas. APMIS 107(11), 1043–1050.PubMedCrossRefGoogle Scholar
  9. 9.
    Xerri, L., Palmerini, F., Devilard, E., Defrance, T., Bouabdallah, R., Hassoun, J., and Birg, F. (2000) Frequent nuclear localization of ICAD and cytoplasmic co-expression of caspase-8 and caspase-3 in human lymphomas. J. Pathol. 192, 194–202.PubMedCrossRefGoogle Scholar
  10. 10.
    Krajewski, S., Krajewska, M., Ellerby, L., Welsh, K., Xie, Z., Deveraux, Q., et al. (1999) Release of caspase-9 from mitochondria during neuronal apoptosis and cerebral ischemia. Proc. Natl. Acad. Sci. USA 96, 5752–5757.PubMedCrossRefGoogle Scholar
  11. 11.
    Krupinski, J., Lopez, E., Marti, E., and Ferrer, I. (2000) Expression of caspases and their substrates in the rat model of focal cerebral ischemia. Neurobio. Dis. 7, 332–342.CrossRefGoogle Scholar
  12. 12.
    Tanaka, M., Momoi, T., and Marunouchi, T. (2000) In situ detection of activated caspase-3 in apoptotic granule neurons in the developiing cerebellum in slice cultures and in vivo. Brain Res. Dev. Brain Res. 121(2), 223–228.PubMedCrossRefGoogle Scholar
  13. 13.
    Hartmann, A., Hunot, S., Michel, P., Muriel, P., Vyas, S., Faucheux, B., et al. (2000) Casapse-3: a vulnerability factor and final effector in apoptotic death of dopaminergic neurons in Parkinson’s disease. ProcNatl. Acad. Sci. USA 97(6), 2875–2880.CrossRefGoogle Scholar
  14. 14.
    Urase, K., Fujita, E., Miho, Y., Kouroku, Y., Mukasa, T., Yagi, Y., et al. (1998) Detection of activated caspase-3 (CPP32) in the vertebrate nervous system during development by a cleavage site-directed antiserum. Brain Res. Dev. Brain Res. 111(1), 77–87.PubMedCrossRefGoogle Scholar
  15. 15.
    Pompeiano, M., Blaschke, A. J., Flavell, R. A., Srinivasan, A., and Chun, J. (2000) Decreased apoptosis in proliferative and postmitotic regions of the Caspase 3-deficient embryonic central nervous system. J. Comp. Neurol. 423(1), 1–12.PubMedCrossRefGoogle Scholar
  16. 16.
    Mukasa, T., Momoi, T., and Momoi, M. Y. (1999) Activation of caspase-3 apoptotic pathways in skeletal muscle fibers in laminin alpha2-defiecient mice. Biochem. Biophys. Res. Commun. 260(1), 139–142.PubMedCrossRefGoogle Scholar
  17. 17.
    Lorz, C., Ortiz, A., Justo, P., Gonzzalez-Cuadrado, S., Duque, N., Gomez-Guerrero, C., and Egido, J. (2000) Proapoptotic fas ligand is expressed by normal kidney tubular epithelium and injured glomeruli. J. Am. Soc. Nephrol. 11, 1266–1277.PubMedGoogle Scholar
  18. 18.
    Basolo, F., Fiore, L., Baldanzi, A., Giannini, R., Dell-Omodarme, M., Fontanini, G., et al. (2000) Suppression of Fas expression and down-regulation of Fas ligand in highly aggressive human thyroid carcinoma. Lab. Invest. 80(9), 1413–1419.PubMedGoogle Scholar
  19. 19.
    Herrnring, C., Reimer, T., Jeschke, U., Makovitzky, J., Kruger, K., et al. (2000) Expression of the apoptosis-inducing ligands FasL and TRAIL in malignant and benign human breast tumors. Histochem. Cell Biol. 113, 189–194.PubMedCrossRefGoogle Scholar
  20. 20.
    Ito, Y., Takeda, T., Sasaki, Y., Sakon, M., Yamada, T., Ishiguro, S., et al. (2000) Expression of fas and fas ligand reflects the biological characteristics but not the status of apoptosis of intrahepatic cholangiocellular carcinoma. Int. J. Mol. Med. 6(4), 581–586.PubMedGoogle Scholar
  21. 21.
    Nakamura, M., Rieger, J., Weller, M., Kim, J., Kleihues, P., and Ohgaki, H. (2000) APO2L/TRAIL expression in brain tumors. Acta Neuropathol. 99, 1–6.PubMedCrossRefGoogle Scholar
  22. 22.
    Srinivas, G., Kusumakumary, P., Nair, M. K., Panicker, K. R., and Pillai, M. R. (2000) Mutant p53 protein, Bcl-2/Bax ratios and apoptosis in paediatric acute lymphoblastic leukaemia. J. Cancer Res. Clin. Oncol. 126(1), 62–67.PubMedCrossRefGoogle Scholar
  23. 23.
    Feuerhake, F., Sigg, W., Hofter, E. A., Dimpfl, T., Welsch, U. (2000) ImMu-nohistochemical analysis of Bcl-2 and Bax expression in relation to cell turnover and epithelial differentiation markers in the non-lactating human mammary gland epithelium. Cell Tissue Res. 299, 47–58.PubMedCrossRefGoogle Scholar
  24. 24.
    Leiter, U., Schmid, R., Kaskel, P., Peter, R., and Krahn, G. (2000) Antiapoptotic bcl-2 and bcl-xL in advanced malignant melanoma. Arch. Dermatol. Res. 292, 225–232.PubMedCrossRefGoogle Scholar
  25. 25.
    Le, M. G., Mathieu, M. C., Douc-Rasy, S., Le Bihan, M. L., Adb El All, H., Spielmann, M., and Riou, G. (1999) c-myc, p53 and bcl-2, apoptosis-related genes in infiltrating breast carcinomas: evidence of a link between bcl-2 pro-tein over-expression and a lower risk of metastasis and death in operable patients. Int. J. Cancer 84(6), 562–567.PubMedCrossRefGoogle Scholar
  26. 26.
    Bukholm, I. and Nesland, J. (2000) Protein expression of p53, p21 (WAF1/ CIP1), bcl-2, Bax, cyclin D1 and pRb in human colon carcinomas. Virchows Arch. 436, 224–288.PubMedCrossRefGoogle Scholar
  27. 27.
    Martin, S. J., Reutelingsperger, C. P., McGahon, A. J., Rader, J. A., van Schie, R. C., LaFace, D. M., and Green, D. R. (1995) Early redistribution of plasma membrane phosphatidyl serine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J. Exp. Med. 182(5), 1545–1556.PubMedCrossRefGoogle Scholar
  28. 28.
    Martin, S. J., Finucane, D.M., Amarante-Mendes, G. P., O’Brien, G. A., and Green, D. R. (1996) Phosphatidylserine externalization during CD95-induced apoptosis of cells and cytoplasts requires ICE/CED-3 protease activity. J. Biol. Chem. 271(46), 28,753–28,756.PubMedCrossRefGoogle Scholar
  29. 29.
    Bronckers, A. L. J. J., Goei, S. W., Dumont, E., Lyaruu, D. M., Woltgens, J. H. M., van Heerde, W. L., et al. (2000) In situ detection of apoptosis in dental and periodontal tissues of the adult mouse using annexin-V-biotin. Histochem. Cell Biol. 113, 293–201.PubMedGoogle Scholar
  30. 30.
    Carr, N. J. (2000) M30 expression demonstrates apoptotic cells correlates with in situ end-labeling, and is associated with Ki-67 expression in large intestinal neoplasms. Arch. Pathol. Lab. Med. 124(12), 1768:1772.PubMedGoogle Scholar
  31. 31.
    Leers, M. P., Kolgen, W., Bjorklund, V., Bergman, T., Tribbick, G., Persson, B., et al. (1999) ImMunocytochmical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis. J. Pathol. 187(5), 567–572PubMedCrossRefGoogle Scholar
  32. 32.
    Mandir, A. S., Przedborski, S., Jackson-Lewis, V., Wang, A., Simbulan-Rosenthal, C. M., Smulson, M. E., et al. (1999) Poly(ADP-ribose) polymerase activation mediates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism. Proc. Natl. Acad. Sci. USA 96, 5774–5779.PubMedCrossRefGoogle Scholar
  33. 33.
    Davis, R. E., Mysore, V., Browning, J. C., Hsieh, J. C., Lu, Q., and Katsikis, P. D. (1998) In situ staining for poly(ADP-ribose) polymerase activity using an NAD analogue. J. Histochem. Cytochem. 46(11), 1279–1289.PubMedGoogle Scholar
  34. 34.
    Hytiroglou, P., Choi, S. W., Theise, N. D., Chaudhary, N., Worman, H. J., and Thung, S. N. (1993) The expression of nuclear lamins in human liver: an immunohistochemical study. Hum. Pathol. 24(2), 169–172.PubMedCrossRefGoogle Scholar
  35. 35.
    Hashimoto, S., Koji, T., Niu, J., Kanematsu, T., and Nakane, P. K. (1995) Differential staining of DNA strand breaks in dying cells by non-radioactive in situ nick translation. Arch. Histol. Cytol. 58(2), 161–170.PubMedCrossRefGoogle Scholar
  36. 36.
    Chun, J. (1998) Detection of cells undergoing Programmed cell death using in situ end-labeling plus, in Apoptosis Detection and Assay Methods (Zhu, L. and Chun, J. M., eds.), Biotechniques Books, Natick, MA, pp. 7–14.Google Scholar
  37. 37.
    Wijsman, J. H., Jonker, R. R., Keijzer, R., van de Velde, C. J., Cornelisse, C. J., and van Dierendonck, J. H. (1993) A new method to detect apoptosis in paraffin sections: in situ end labeling of fragmented DNA. J. Histochem. Cytochem. 41(1), 7–12.PubMedGoogle Scholar
  38. 38.
    Wood, K. A., Diapsquale, B., and Youle, R. J. (1993) In situ labeling of gran-ule cells for apoptosis-associated DNA fragmentation reveals different mecha-nisms of cell loss in developing cerebellum. Neuron. 11(4), (Zhu, L. and Chun, J.M., eds.) 621–632.Google Scholar
  39. 39.
    Gavrieli, Y., Sherman, Y., and Ben-Sasson, S. A. (1992) Identification of pro-gramMed cell death in situ via specific labeling of nuclear DNA fragmentation. J. CellBiol. 119(3), 493–501.CrossRefGoogle Scholar
  40. 40.
    Wheeldon, E. B., Williams, S. M., Soanes, A. R., James, N. H., Roberts, R. A. (1995) Quantitation of apoptotic bodies in rat liver by in situ end labelling (ISEL): Correlation with morphology. Toxicol. Pathol. 23(3), 410–415.PubMedCrossRefGoogle Scholar
  41. 41.
    Frankfurt, O. S., Robb, J. A., Sugarbaker, E. V., and Villa, L. (1997) Apoptosis in breast carcinomas detected with monoclonal antibody to single-stranded DNA: relation to bcl-2 expression, hormone receptors, and lymph node me-tastases. Clin. Cancer Res. 3(3), 465–471.PubMedGoogle Scholar
  42. 42.
    Frankfurt, O. S., Robb, J. A., Sugarbaker, E. V., and Villa, L. (1996) Mono-clonal antibody to single-stranded DNA is a specific and sensitive cellular marker of apoptosis. Exp. Cell Res. 226(2), 387–397.PubMedCrossRefGoogle Scholar
  43. 43.
    Frankfurt, O. S. (1998) Detection of apoptotic cells with monoclonal antibod-ies to single-stranded DNA, in Apoptosis Detection and Assay Methods (Zhu, L. and Chun, J. M., eds.) Biotechniques Books, Natick, MA, pp.47–62.Google Scholar
  44. 44.
    Stinchcombe, S., Buchmann, A., Bock, K. E., and Schwarz, M. (1995) Inhibi-tion of apoptosis during 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated tumour promotion in rat liver. Carcinogenesis 16(6), 1271–1275.PubMedCrossRefGoogle Scholar
  45. 45.
    Kong, J., and Ringer, D. P. (1995) Quantitative in situ image analysis of apoptosis in well and poorly differentiated tumor from rat liver. Am. J. Pathol. 147(6), 1626–1632.PubMedGoogle Scholar
  46. 46.
    Fujita, K., Ohyama, H., and Yamada, T. (1997) Quantitative comparison of in situ methods for detecting apoptosis induced by X-ray irradiation in mouse thymus. Histochem. J. 29, 823–830.PubMedCrossRefGoogle Scholar
  47. 47.
    Bursch, W., Patte, S., Putz, B., Barthel, G., and Schulte-Hermann, R. (1990) Determination of the length of the histological stages of apoptosis in normal liver and in altered hepatic foci of rats. Carcinogenesis 11(5), 847–853.PubMedCrossRefGoogle Scholar
  48. 48.
    Moolgavkar, S. H., Luebeck, E. G. (1992) Interpretation of labeling indices in the presence of cell death. Carcinogenesis 13(6), 1007–1010.PubMedCrossRefGoogle Scholar
  49. 49.
    Howard, C. V., Reed, M. G. (1998) Unbiased Stereology: Three Dimensional Measurement in Stereology. Springer-Verlag, New York, New York.Google Scholar
  50. 50.
    Kerr, J. F. R., Wyllie, A. H., and Currie, A. R. (1972) Apoptosis: A basic bio-logical phenomenon with wide-ranging implications in tissue kinetics. Brt. J. Cancer. 68, 239–257.CrossRefGoogle Scholar
  51. 51.
    Angermuller, S., Schuman, J., Fahimi, H. D., Tiege, G. (1999) Ultrastructural alterations of mitochondria in pre-apoptotic and apoptotic hepatocytes of TNF-alpha-treated galactosamine-sensitized mice. Ann NYAcad. Sci. 887, 12–1748.CrossRefGoogle Scholar
  52. 52.
    Payne C. M., and Cromey, D. W. (1991) Ultrastructural analysis of apoptotic and normal cells using digital imaging techniques. J. Comp.-Assisted Micros-copy 3 (1), 33–50.Google Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Matthew A. Wallig
    • 1
  • Curtis M. Chan
    • 2
  • Nancy A. Gillett
    • 2
  1. 1.Department of Veterinary PathobiologyUniversity of Illinois at Urbana-ChampaignUrbana
  2. 2.A Division of Charles River Laboratories, Inc.Sierra BiomedicalSparks

Personalised recommendations