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Direct immunoenzyme double staining applicable for monoclonal antibodies

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Summary

A novel method for immunoenzymatic double staining was developed, using primary antibodies directly labeled with either horseradish peroxidase or alkaline phosphatase. The enzyme-antibody conjugates were applied simultaneously on sections of human tonsil. Intracytoplasmic antigens like immunoglobulins and light chains could easily be detected simultaneously in the same tissue section. With antibodies against cell surface antigens like IgM and T cell antigens areas containing B and T cells could be clearly distinghuished. This method opens the possibility to perform double staining with two monoclonal antibodies.

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References

  • Avrameas S (1969) Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies. Immunochemistry 6:43–53

    Google Scholar 

  • Avrameas S, Ternynck T (1971) Peroxidase labeled antibody and Fab conjugates with enhanced intracellular penetration. Immunochemistry 8:1175–1179

    Google Scholar 

  • Boorsma DM (1983) Preparation of HRP-labelled antibodies. In: Cuello AC (ed) Immunohistchemistry. IBRO Handbook. John Wiley and Sons, Chichester, UK, pp 87–100

    Google Scholar 

  • Boorsma DM, Kalsbeek GL (1975) A comparative study of horseradish peroxidase conjugates prepared with a one-step and two-step method. J. Histochem Cytochem 23:200–207

    Google Scholar 

  • Boorsma DM, Streefkerk JG (1976) Peroxidase conjugate chromatography: Isolation of conjugates prepared with glutaraldehyde or periodate using polyacrylamide-agarose gel. J Histochem Cytochem 24:481–486

    Google Scholar 

  • Boorsma DM, Streefkerk JG (1979) Periodate or glutaraldehyde for preparing peroxidase conjugates? J Immunol Methods 30:245–255

    Google Scholar 

  • Boorsma DM, Cuello AC, VanLeeuwen FW (1982) Direct immunocytochemistry with a horseradish peroxidase-conjugated monoclonal antibody against substance P. J Histochem Cytochem 30:1211–1216

    Google Scholar 

  • Bosman FT, Lindeman J, Kuiper G, VanderWal A, Keuning J (1977) The influence of fixation on immunoperoxidase staining of plasmacells in paraffin sections of intestinal biopsy specimens. Histochemistry 53:57–62

    Google Scholar 

  • Campbell GT, Bhatnagar AS (1976) Simultaneous visualization by light microscopy of two pituitary hormones in an single tissue section using a combination of indirect immunohistochemical methods. J Histochem Cytochem 24:448–452

    Google Scholar 

  • Ford DJ, Radin R, Pesce AJ (1978) Characterization of glutaraldehyde coupled alkaline phosphatase-antibody and lactoperoxidase-antibody conjugates. Immunochemistry 15:237–243

    Google Scholar 

  • Hsu SM, Soban E (1982) Color modification of diaminobenzidine (DAB) precipitation by metallic ions and its application for double immunohistochemistry. J Histochem Cytochem 30:1079–1082

    Google Scholar 

  • Mason DY, Sammons R (1978) Alkaline phosphatase and peroxidase for double immunoenzymatic labeling of cellular constituents. J Clin Pathol 31:454–460

    Google Scholar 

  • Nakane PK (1968) Simultaneous localization of multiple tissue antigens using the peroxidase-labeled antibody method: a study on pituitary glands of the rat. J Histochem Cytochem 16:557–560

    Google Scholar 

  • Sternberger LA, Joseph SA (1979) The unlabeled antibody method. Contrasting color staining of paired pituitary hormones without antibody removal. J Histochem Cytochem 27:1424–1429

    Google Scholar 

  • Streefkerk JG (1972) Inhibition of erythrocyte pseudoperoxidase activity by treatment with hydrogen peroxide following methanol. J Histochem Cytochem 20:829–831

    Google Scholar 

  • Suffin SC, Muck KB, Young JC, Lewin K, Porter DD (1979) Improvement of the glucose oxidase immunoenzyme technic. Use of a tetrazolium whose formazan is stable without heavy metal chelation. Am J Clin Pathol 71:492–496

    Google Scholar 

  • Tax WJM, Willems HW, Kibbelaar MDA, de Groot J, Capel PJA, de Waal RMW, Reekers P, Koene RAP (1981) Monoclonal antibodies against human thymocytes and lymphocytes, 29th Protides of the biological fluids. Pergamon Press, Oxford, UK, pp 701–704

    Google Scholar 

  • Tramu G, Pillez A, Leonardelli J (1978) An efficient method of antibody elution for the successive or simultaneous localization of two antigens by immunocytochemistry. J Histochem Cytochem 26:322–324

    Google Scholar 

  • Valnes K, Brandtzaeg P (1982) Comparison of paired immunofluorescence and paired immunoenzyme staining methods based on primary antisera from the same species. J Histochem Cytochem 30:518–523

    Google Scholar 

  • Van de Sande F, Dierickx K (1976) Immunocytochemical demonstration of separate vasotocinergic and mesotocinergic neurons in the amphibian hypothalamic magnocellular neurosecretory system. Cell Tissue Res 175:284–289

    Google Scholar 

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Authors and Affiliations

  1. Immunocytochemistry Unit, Department of Dermatology Academic Hospital, Faculty of Medicine, Free University, NL-1081 HV, Amsterdam, The Netherlands

    D. M. Boorsma

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  1. D. M. Boorsma
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Boorsma, D.M. Direct immunoenzyme double staining applicable for monoclonal antibodies. Histochemistry 80, 103–106 (1984). https://doi.org/10.1007/BF00679982

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  • DOI: https://doi.org/10.1007/BF00679982

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