Summary
The denaturing effects of various types of fixative solutions on 5 cell surface antigens on mouse T-lymphocytes (Thy-1, T-200, Lyt-1, Lyt-2, and Th-B) were studied. For this purpose, cells were fixed with paraformaldehyde, glutaraldehyde, acrolein and osmium tetroxide at various concentrations. Fixed cells were then incubated with monoclonal antibodies and appropriate second stage antibodies or conjugates. The degree of antibody binding to these cells was determined quantitatively using flow-cytometry with a fluorescence-activated cell sorter or with a semi-automatic micro-ELISA system. The data obtained indicate that paraformaldehyde and glutaraldehyde preserve all five tested antigen molecules, whereas antibody binding to cells fixed in acrolein and osmium tetroxide is rapidly reduced at increasing concentrations of the fixative. The optimal concentration of paraformaldehyde is in the range 0.5–1%, whereas glutaraldehyde should be used at concentrations between. 0.05 and 0.1%. Cells fixed with 0.5% paraformaldehyde or with 0.05% glutaraldehyde are stable and can be stored for at least one week prior to incubation with antibodies.
Similar content being viewed by others
References
Bullock, G. R. &Petrusz, P. (1982)Techniques in Immunocytochemistry, Vol. 1. New York, London: Academic Press.
Collins, J. S. &Goldsmith, T. H. (1981) Spectral properties of fluorescence induced by glutaraldehyde fixation.J. Histochem. Cytochem. 29, 411–4.
Curran, R. C. &Gregory, J. (1978) Demonstration of immunoglobulin in cryostat and paraffin sections of human tonsil by immunofluorescence and immunoperoxidase techniques.J. clin. Path. 31, 974–83.
De Josselin de Jong, J. E., Jongkind, J. F. &Ywema, H. R. (1980) A scanning inverted microfluorometer with electronic shutter control for automatic measurements in micro-test plates.Analyt. Biochem. 102, 120.
Hopwood, D. (1972) Theoretical and practical aspects of glutaraldehyde fixation.Histochem. J. 4, 267–303.
Howlett, B. J., Vithanage, H. I. M. V. &Knox, R. B. (1981) Immunofluorescence localization of two water soluble glycoproteins including, the major allergen from the pollen of ryegrass,Lolium perenne.Histochem. J. 13, 461–80.
Judd, M. A. &Britten, K. J. M. (1982) Tissue preparation for the demonstration, of surface antigens by immunoperoxidase techniques.Histochem. J. 14, 747–53.
Kraehenbuehl, J. P., De, Grandi, P. B. &Campiche, M. A. (1971) Ultrastructural localisation of intracellular antigens using enzyme labelled antibody fragments.J. Cell. Biol. 50, 432–45.
Ledbetter, J. A. &Herzenberg, L. A. (1979) Xenogeneic monoclonal antibodies to mouse lymphoid differentiation antigens.Immun. Rev. 47, 63–90.
Mclean, J. W. &Nakane, P. K. (1974) Periodate-lysine paraformaldehyde fixative. A new fixative for immunoelectronmicroscopy.J. Histochem. Cytochem. 22, 1077–83.
Mcmillan, E. M., Wasik, R. &Everett, M. A. (1982) OKT-6 positive cells. Their demonstration in human thymus and the effect of fixation of the immunoperoxidase reactionArchs. Path. Lab. Med. 106, 9–12.
Nishiya, K., Chiao, J. W. &De Sousa, M. (1980) Iron binding proteins in selected human peripheral blood cell sets: Immunofluorescence.Br., J. Haemat.,46, 235–45.
Reitamo, S., Ranki, A., Konttinen, Y. T. &Hairy, P. (1980) Immunoperoxidase identification of intracellular immunoglobulins from cell smears.Am. J. clin. Path. 73, 249–49.
Smit, J. W., Meijer, C. J. L. M., Decary, F. &Feltkamp-Vroom, Th. (1974) Paraformaldehyde fixation in immunofluorescence and immunoelectronmicroscopy. Preservation of tissue and cell surface antigens.J. Immun. Meth. 6, 93–9.
Stein, H., Bonk, A., Tolksdorf, G., Lennert, K., Rodt, H. &Gerdes, J. (1980) Immunohistologic analysis of the organisation of normal lymphoid tissue and non-Hodgkin lymphoma's.J. Histochem. Cytochem. 28, 746–60.
Stoeckenius, W. &Mahr, S. C. (1965) Studies on the reaction of osmium tetroxide and related compounds.Lab. Invest. 14, 29–41.
Van, Ewijk, W., Coffman, R. C. &Weissman, I. L. (1980) Immunoelectronmicroscopy of cell surface antigens: a quantitative analysis of antibody binding after different fixation protocols.Histochem. J. 12, 349–61.
Van Ewijk, W., Van Soest, P. L. &Van den Engh, G. J. (1981) Fluorescence analysis and anatomic distribution of mouse T lymphocyte subsets defined by monoclonal antigens to the antigens Thy-1, Lyt-1, Lyt-2 and T-200.J. Immunol. 127, 2595–604.
Van Soest, P. L., De Josselin de Jong, J. E. Lansdorp, P. M. &Van Ewijk, W. (1984) An automatic fluorescence micro-ELISA system for quantitative screening of hybridoma supernatants using a protein-A-β-galactosidase conjugate.Histochem. J. 16, 21–35.
Webb, J. D. &Dorling, J. (1972) The use of peroxidase labelled antiglobulin, for ultrastructural localisation of tissue antigens reacting with serum antibodies.J. Immun Meth. 2, 145–57.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
van Ewijk, W., van Soest, P.L., Verkerk, A. et al. Loss of antibody binding to prefixed cells: Fixation parameters for immunocytochemistry. Histochem J 16, 179–193 (1984). https://doi.org/10.1007/BF01003548
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01003548