Summary
Combined application of a non-radioactivein situ DNA hybridization procedure and the immunoperoxidase technique on one tissue section is described. Of six potential protocols, only one proved to be successful. First, the immunohistochemical procedure including visualization of enzyme activity is performed; thein situ DNA hybridization protocol is then applied. Using this protocol, several antigens, detected with monoclonal antibodies, and target DNAs, detected by using biotinylated human cytomegalovirus or human papilloma virus type 16 DNA probes, could be distinguished by their peroxidase activity (brown precipitate) and alkaline phosphatase activity (purple—blue precipitate) respectively. The method allows immunophenotyping of virus-infected cells as well as simultaneous visualization of two viral parameters. This technique has important implications for research and diagnostic purposes.
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BOORSMA, D. M. (1984) Direct immunoenzyme double staining applicable for monoclonal antibodies.Histochemistry 80, 103–6.
BRAHIC, M., HAASE, A. T. & CASH, E. (1984) Simultaneousin situ detection of viral RNA and antigens.Proc. Natl. Acad. Sci. USA 81, 5445–8.
DE JONG, A. S. H., VAN KESSEL-VAN VARK, M. & RAAP, A. K. (1985) Sensitivity of various visualization methods for peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry.Histochem. J. 17, 1119–30.
GRAHAM, R. C. & KARNOFSKY, M. J. (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse: ultrastructural cytochemistry by a new technique.J. Histochem. Cytochem. 14, 291–302.
HOEFLER, H., CHILDERS, H., MOTMINY, M. R., LECHAN, R. M., GOODMAN, R. H. & WOLFE, H. J. (1986)In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes.Histochem. J. 18, 597–604.
HOUTHOFF, H. J., VOLKERS, H. H., VAN DEN BERG, F. M., VAN DER LOOS, C. M. & LEUNISSE, J. (1988) Detection of cytomegalovirus DNA and antigenic expression. Comparison of sensitivity and selectivity.Proc. 8th Int. Congr. Histochem. Cytochem. 36, 947 (Abstr.).
KOENIG, S., GENDELMAN, H. E., ORENSTEIN, J. M., DAL CANTO, M. C., PEZESHKPOUR, G. H., YUNGBLUTH, M., JANOTTA, F., AKSAMIT, A., MARTIN, M. A. & FANCI, A. S. (1986) Detection of AIDS virus in macrophages in brain tissue from AIDS patients with encephalopathy.Science 233, 1089–93.
LANGER, P. R., WALDORP, A. A. & WARD, D. C. (1981) Enzymatic synthesis of biotin labeled polynucleotides: novel nuclei acid affinity probes.Proc. Natl. Acad. Sci. USA 73, 6633–7.
LEARY, J. J., BRIGATI, D. J. & WARD, D. C. (1983) Rapid and sensitive colorimetric method for visualizing biotinlabeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots.Proc. Natl. Acad. Sci. USA 80, 4045–9.
LONING, T., MILDE, K. & FOSS, H. D. (1986)In situ hybridization for the detection of cytomegalovirus (CMV) infection. Application of biotinylated CMV—DNA probes on paraffin embedded specimen.Virchows Arch. 409, 777–90.
MASON, D. Y. & GATTER, K. C. (1987) The role of immunocytochemistry in diagnostic pathology.J. Clin. Pathol. 40, 1042–54.
RAAP, A. K., GEELEN, J. L., VAN DER MEER, J. W. M., VAN DE RIJKE, F. M., VAN DEN BOOGAART, P. & VAN DER PLOEG, M. (1988) Non-radioactivein situ hybridization for the detection of cytomegalovirus infections.Histochemistry 88, 367–73.
SHIVERS, B. D., HARLAN, R. E., PFAFF, D. W. & SCHACHTER, B. S. (1986) Combination of immunocytochemistry andin situ hybridization in the same tissue section of rat pituitary.J. Histochem. Cytochem. 34, 39–43.
STERNBERGER, L. A. & JOSEPH, S. A. (1979) The unlabeled antibody method: contrasting color staining of paired pituitary hormones without antibody removal.J. Histochem. Cytochem. 27, 1424–9.
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, 518–24.
VALNES, K. & BRANDTZAEG, P. (1984) Paired indirect immunoenzyme staining with primary antibodies from the same species. Application of horseradish peroxidase and alkaline phosphatase as sequential labels.Histochem. J. 16, 477–87.
VAN DEN BERG, F. M., JIWA, M., ROOK, R. & GEELEN, J. L. M. C. (1988) Analysis and isolation of cytomegalovirus DNA by field inversion gel electrophoresis.J. Gen. Virol. 69, 699–704.
VAN DEN BERG, F. M., SCHIPPER, M. E. I., JIWA, M., ROOK, R., VAN DE RIJKE, F. M. & TIGGES, A. J. (1989) Implausibility of an aetiological association between cytomegalovirus and Kaposi's sarcoma shown by four techniques.J. Clin. Pathol. 42, 128–31.
VAN DER LOOS, C. M., DAS, P. K. & HOUTHOFF, H. J. (1987) An immunoenzyme triple-staining method using both polyclonal and monoclonal antibodies from the same species. Application of combined direct, indirect and avidin—biotin complex (ABC) technique.J. Histochem. Cytochem. 35, 1199–204.
VAN DER LOOS, C. M., DAS, P. K., VAN DEN OORD, J. J. & HOUTHOFF, H. J. (1989) Multiple immunoenzyme staining techniques. Use of fluoresceinated, biotinylated and unlabeled monoclonal antibodies.J. Immunol. Methods,117, 45–52.
VAN DER LOOS, C. M., VAN DEN OORD, J. J., DAS, P. K. & HOUTHOFF, H. J. (1988) Use of commercially available monoclonal antibodies for immunoenzyme double staining.Histochem. J. 20, 409–13.
VANDESANDE, F. (1983) Immunohistochemical double staining techniques. InImmunochemistry, IBRO Handbook (edited by CUELLO, A. C.), pp. 257–72. Chichester: John Wiley and Sons.
VOLKERS, H. H. (1988) Microwave irradiation and DNAin situ hybridization. InMicrowave Cookbook of Pathology, 2nd edn (edited by BOON, M. E. & KOK, L. P.), pp. 216–19. Leiden: Coulomb Press.
WOLBER, R. A. & LLOYD, R. C. (1988) Cytomegalovirus detection by non-isotopicin situ DNA hybridization and viral antigen immunostaining using a two-color technique.Hum. Pathol. 19, 736–41.
WOLFSON, B., MANNING, R. W., DAVIS, L. G., ARENTZEN, R. & BALDINO, F. (1985) Co-localization of corticotropin factor and vasopressin mRNA in neurones after adrenalectomy.Nature 351, 59–61.
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Van Der Loos, C.M., Volkers, H.H., Rook, R. et al. Simultaneous application ofin situ DNA hybridization and immunohistochemistry on one tissue section. Histochem J 21, 279–284 (1989). https://doi.org/10.1007/BF01757180
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DOI: https://doi.org/10.1007/BF01757180