Summary
A DNA-in situ hybridization protocol was adapted for application to sections of routinely processed paraffin embedded material. This protocol was developed previously for detecting DNA-virus infected cells in whole cell preparations and employs biotinylated DNA as probe. Three different biotin detection methods were optimized and applied. The first uses streptavidin and a biotinylated complex of alkaline phosphatase, the second consists of an immunogold-silver staining, and the third of a peroxidase technique using a silver amplification. The alkaline phosphatase method was the most rapid, and as sensitive as the immunogold-silver staining. The peroxidase method was the most sensitive. Microwave irradiation was applied to the different incubation steps of these three detection methods. Short incubations with microwave irradiation gave very poor results when peroxidase labelled antibodies were used. Short incubation with microwave irradiation gave results comparable to those obtained with conventional incubations, when streptavidin, antibiotin, complexed alkaline phosphatase, or gold labelled goat antirabbit were used. It was thus shown that microwave irradiation creates the possibility of a very rapid label-detection for nonradioactive DNA-in situ hybridization.
Similar content being viewed by others
Refrrences
Birrel, G. B., Habliston, D. L., Hedberg, K. K. &Griffith, O. H. (1986) Silver-enhanced colloidal gold as a cell surface marker for photoelectron microscopy.J. Histochem. Cytochem. 34, 339–45.
Boon, M. E. &Kok, L. P. (1988) Microwave Ovens. InMicrowave cookbook of pathology: The art of microscopic visualization. 2nd edn. pp. 42–72. Leiden, The Netherlands: Coulomb Press Leyden.
Boon, M. E., Kok, L. P., Moorslag, H. E. &Suurmeijer, A. J. H. (1989) Accelerated immunogold-silver and immunoperoxidase staining of paraffin sections with use of microwave irradiation.Am. J. Clin. Pathol. 91, 137–43.
Brigati, D. J., Myerson, D., Leary, J. J., Spalholz, B., Travis, S. Z., Fong, C. K. Y., Hsiung, G. D. &Ward, D. C. (1983) Detection of viral genomes in cultured cells and paraffin embedded tissue sections using biotinla-belled hybridization probes.Virology. 126, 32–50.
Burns, J., Chan, V. T. W., Jonasson, J. A., Fleming, K. A., Taylor, S. &McGee, J. O'D. (1985) Sensitive system for visualizing biotinylated DNA probes hybridizedin situ.J. Clin. Pathol. 38, 1085–92.
Burns, J., Graham, A. K., Frank, C., Fleming, K. A., Evans, M. F. &McGee, J. O'D. (1987) Detection of low copy human papilloma virus DNA and RNA in routine paraffin sections of cervix by non-isotopicin situ hybridization.J. Clin. Pathol. 40, 865–9.
Dartmann, K., Schwarz, E., Gissmann, L. &Zur Hausen, H. (1986) The neuclotide sequence and genome organisation of human papilloma virus type II.Virlogy.151, 124–30.
De Villiers, E. M., Gissmann, L. &Zur Hausen, H. (1981) Molecular cloning of viral DNA from human genital warts.J. Virol. 40, 932–5.
De Waele, M., Renmans, W., Segers, E., Jochmans, K. &An Camp, B. (1988) Sensitive detection of immunogold-silverstaining with darkfield and epipolarization microscopy.J. Histochem. Cytochem. 36, 670–83.
Gissmann, L., Wolnik, L., Ikenberg, H., Koldovsky, U., Schnurch, H. G. &Zur Hausen, H. (1983) Human papilloma virus types 6 and 11 DNA sequences in genital and laryngeal papillomas and in some cervical cancers.Proc. Natl. Acad. Sci. USA 80, 560–3.
Hjerpe, A., Boon, M. E., &Kok, L. P. (1988) Microwave stimulation of an immunological reaction (CEA-anti CEA) and its use in immunohistochemistry.Histochem. J. 20, 388–96.
Hopman, A. H. N., Wiegant, J. &Van Duijn, P. (1986) A new hybridocytochemical method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands.Histochem. 84, 169–78.
Landegent, J. E., Jansen in De Wal, N., Baan, R. A., Hoeijm, J. H. J. &Van Der Ploeg, M. (1984). 2-Acetylaminofluorene-modified probes for the indirect hybridocytochemical detection of specific nucleic acid sequences.Exp. Cell Res. 153, 61–72.
Langer, P. R., Waldrop, A. A. &Ward, D. C. (1981) Enzymatic synthesis of biotin-labelled polynucleotides: novel nucleic acid affinity probes.Proc. Natl. Acad. Sci. USA 78, 6633–7.
Leong, A. S. Y. &Milios, J. (1986) Rapid immunoperoxidase staining of lymphocyte antigens using microwave irradiation.J. Pathol. 148, 183–7.
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. (1987) Nonradioactivein situ hybridization for the detection of cytomegalovirus infectionsHistochem. 88, 367–73.
Scopsi, L. &Larsson, L. I. (1985) Increased sensitivity in immunocytochemistry. Effects of double application of antibodies and of silver intensification on immunogold and peroxidase-antiperoxidase staining techniques.Hitochem. 82, 32–9.
Thomas, N., Bennett, R. &Jones, C. N. (1987) A comparison of immunocytochemical enhancement methods using rapid microtitre immunocytochemistry assay (MIA).J. Immunol. Methods 104, 201–7.
Van De Kant, H. J. G., Boon, M. E. &De Rooij (1988) Microwave-aided technique to detect bromodeoxyuridine in S-phase cells using immunogold-silver staining and plastic-embedded sections.Histochem. J. 20, 335–40.
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., Jiwa, M. Rook, R., Van De Rijke, F. &Tigges, B. (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. &Tigges, A. J. (1988) Three step indirect peroxidase method for frozen sections. InMicrowave cookbook of pathology: The art of microscopic visualisation. 2nd edn. edited byBoon &Kok) pp. 171–3. Leiden, The Netherlands: Coulomb Press Leyden.
Walboomers, J. &Ter Schegget, J. (1976) A new method for the isolation of herpes simplex virus type 2 DNA.Virlogy 74, 256–8.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Van Den Brink, W.J., Zijlmans, H.J.M.A.A., Kok, L.P. et al. Microwave irradiation in label-detection for diagnostic DNA-in situ hybridization. Histochem J 22, 327–334 (1990). https://doi.org/10.1007/BF01003165
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01003165