Summary
In contrast to that in paraffin-embedded tissue, the reactivity of monoclonal PCNA antibody PC10 on cryostat sections requires a special fixation procedure as the target epitope is seemingly not accessible to its antibody. A panel of 18 fixation protocols was investigated. Chilled methanol or acetone, or PLP (paraformaldehyde-lysine-periodate) was found to be unsuitable for skin preparations. A two-step fixation protocol was developed for normal skin and basal cell carcinomas. They were fixed first in 3.4% buffered formaldehyde, followed by fixation in 2:1 v/v ethanol-acetic acid. Following this fixation regime, cryostat sections displayed the same PCNA/PC10 labelling pattern as paraffin sections of formalin-fixed tissue.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Berlingin, E., Heenen, M. & Galand, P. (1992) Measurement of S phase duration in human epidermis using cyclin immunostaining and 3H-thymidine pulse labelling. Arch. Dermatol. Res. 284, 238–41.
Bravo, R. & Macdonald-Bravo, H. (1987) Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites. J. Cell Biol. 105, 1549–54.
Celis, J. E. & Celis, A. (1985) Cell cycle-dependent variations in the distribution of the nuclear protein cyclin/proliferating cell nuclear antigen in cultured cells: subdivision of S phase. Proc. Natl Acad. Sci. USA 82, 3262–6.
Dawson, A. E., Norton, J. A. & Weinberg, D. S. (1990) Comparative assessment of proliferation and DNA content in breast carcinoma by image analysis and flow cytometry. Am. J. Pathol. 136, 1115–24.
Diebold, J., Lai, M. D. & Löhrs, U. (1992) Analysis of proliferative activity in colorectal mucosa by immunohistochemical detection of proliferating cell nuclear antigen (PCNA). Methodological aspects and application to routine diagnostical material. Virchows Arch. B Cell Pathol. 62, 283–9.
Furukawa, F., Imamura, S., Fujita, M., Kinoshita, K., Yoshitake, K., Brown, W. R. & Norris, D. A. (1992) Immunohistochemical localization of proliferating cell nuclear antigen/cyclin in human skin. Arch. Dermatol. Res. 284, 86–91.
Galand, P. & Degraef, C. (1989) Cyclin/PCNA immunostaining as an alternative to tritiated thymidine pulse labelling for marking S phase cells in paraffin sections from animal and human tissues. Cell Tissue Kinet. 22, 383–92.
Hall, P. A., Stearn, P. M., Butler, M. G. & D'Ardenne, A. J. (1987) Acetone/periodate-lysine-paraformaldehyde (PLP) fixation and improved morphology of cryostat sections for immunohistochemistry. Histopathology 11, 93–101.
Hall, P. A., Levison, D. A., Woods, A. L., Yu, C. C. W., Kellock, D. B., Watkins, J. A., Barnes, D. M., Gillett, C. E., Camplejohn, R., Dover, R., Waseem, N. H. & Lane, D. P. (1990) Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: an index of cell proliferation with evidence of deregulated expression in some neoplasms. J. Pathol. 162, 285–94.
Kawakita, N., Seki, S., Yanai, A., Sakaguchi, H., Kuroki, T., Mizoguchi, Y., Kobayashi, K. & Monna, T. (1992) Immunocytochemical identification of proliferative hepatocytes using monoclonal antibody to proliferating cell nuclear antigen (PCNA/cyclin). Comparison with immunocytochemical staining for DNA polymerase-alpha. Am. J. Clin. Pathol. 97 (suppl. 1), S14–20.
Kurki, P., Ogata, K. & Tan, E. M. (1988) Monoclonal antibodies to proliferating cell nuclear antigen (PCNA)/cyclin as probes for proliferating cells by immunofluorescence microscopy and flow cytometry. J. Immunol. Meth. 109, 49–59.
McCormick, D. & Hall, P. A. (1992) The complexities of proliferating cell nuclear antigen. Histopathology 21, 591–4.
Miyagawa, S., Okada, N., Takasaki, Y., Iida, T., Kitano, Y., Yoshikawa, K., Sakamoto, K. & Steinberg, M. L. (1989) Expression of proliferating cell nuclear antigen/cyclin in human keratinocytes. J. Invest. Dermatol. 93, 678–81.
Ogata, K., Ogata, Y., Nakamura, R. M. & Tan, E. M. (1985) Purification and N-terminal amino acid sequence of proliferating cell nuclear antigen (PCNA)/cyclin and development of ELISA for anti-PCNA antibodies. J. Immunol. 135, 2623–7.
Pearse, A. G. E. (1980) Histochemistry: Theoretical and Applied. Vol. 1, 4th edn. pp. 97–158. Edinburgh: Churchill Livingstone.
Roos, G., Landberg, G., Huff, J. P., Houghten, R., Takasaki, Y. & Tan, E. M. (1993) Analysis of the epitopes of proliferating cell nuclear antigen recognized by monoclonal antibodies. Lab. Invest. 68, 204–10.
Tsuji, T., Shrestha, P., Yamada, K., Takagi, H., Shinozaki, F., Sasaki, K., Maeda, K. & Mori, M. (1992) Proliferating cell nuclear antigen in malignant and premalignant lesions of epithelial origin in the oral cavity and the skin: an immunohistochemical study. Virchows Arch A Pathol Anat 420, 377–83.
VanDierendonck, J. H., Wijsman, J. H., Keijzer, R., van deVelde, C. J. H. & Cornelisse, C. J. (1991) Cell-cycle-related staining patterns of anti-proliferating cell nuclear antigen monoclonal antibodies. Comparison with BrdUrd labeling and Ki-67 staining. Am. J. Pathol. 138, 1165–72.
Waseem, N. H. & Lane, D. P. (1990) Monoclonal antibody analysis of the proliferating cell nuclear antigen (PCNA). Structural conservation and the detection of a nucleolar form. J. Cell Sci. 96, 121–9.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Baum, HP., Reichrath, J., Theobald, A. et al. Fixation requirements for the immunohistochemical reactivity of PCNA antibody PC10 on cryostat sections. Histochem J 26, 929–933 (1994). https://doi.org/10.1007/BF00174008
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00174008