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Use of commercially available monoclonal antibodies for immunoenzyme double staining

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Summary

An immunoenzyme double-staining method for the simultaneous detection of two cellular epitopes, using commercially available mouse monoclonal antibodies, is described. The method employs a combination of the suppression of endogenous biotin and two successive indirect techniques with a blocking step in between. The first indirect method involves an unlabelled monoclonal antibody followed by an alkaline phosphatase-conjugated goat anti-mouse immunoglobulin. After a blocking step with normal mouse serum, the second indirect method is applied using a biotinylated monoclonal antibody followed by the visualization of this antibody by avidin-biotinylated peroxidase complex (ABC) or rabbit anti-biotin and peroxidase-conjugated swine anti-rabbit immunoglobulin in successive steps. Using these methods in combination with the introduction of dioctyl sodium sulphosuccinate and tetramethylbenzidine as chromogens for peroxidase activity, two cellular epitopes could be distinguished clearly in tissue sections by the green-and violet-stained peroxidase and alkaline phosphatase activities, respectively. The expression of two epitopes on the same cellular constituent is outlined by the coappearance of both enzyme activities as a bluish-purple colour. This method allows for the simultaneous identification, localization and enumeration of two cellular epitopes. These can serve as parameters for a number of pathological processes.

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Author notes

  1. J. J. Van Den Oord

    Present address: Department of Pathology II, University Hospital Sint Rafael, Leuven, Belgium

Authors and Affiliations

  1. Department of Pathology, Academic Medical Centre, Meibergdreef 9, 105 AZ, Amsterdam, The Netherlands

    C. M. Van Der Loos, J. J. Van Den Oord, P. K. Das & H. J. Houthoff

  2. Department of Dermatology, Academic Medical Centre, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands

    P. K. Das

Authors
  1. C. M. Van Der Loos
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  2. J. J. Van Den Oord
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  3. P. K. Das
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  4. H. J. Houthoff
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Van Der Loos, C.M., Van Den Oord, J.J., Das, P.K. et al. Use of commercially available monoclonal antibodies for immunoenzyme double staining. Histochem J 20, 409–413 (1988). https://doi.org/10.1007/BF01002426

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  • DOI: https://doi.org/10.1007/BF01002426

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