Abstract
DNA labeled with non-radioactive markers have been used for in situ hybridization of specific DNA or RNA in cells and tissues. The presence of protruding markers on the probe DNA has been attributed to be the cause for the loss of sensitivity and specificity of hybridization. In search of a non-protruding marker, we investigated the possibility of the use of a T-T dimer which can be generated easily in DNA while being a potent hapten as a marker for DNA [1]. A T-T dimer in DNA was generated by UV irradiation (254 μm). Following hybridization with complementary DNA or RNA in cells and tissues, the T-T dimerized DNA (T-T DNA) was detected immunohistochemically using rabbit anti-T-T DNA and peroxidase-labeled goat anti-rabbit IgG. It was found that the use of T-T as a marker offers several advantages over other markers: it appears not to interfere with hybridization efficiency, is simple to make, and can be detected with high sensitivity.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Nakane PK, Moriuchi T, Koji T, Tanno M, Abe K (1987) In situ localization on mRNA using thymine-thymine dimerized cDNA. Acta Histochem Cytochem 20: 229–243
Takahashi S, Nakane K (1988) Detection of viral genes in the tissues by in situ hybridization. Clin Immunol 20: 172–178
Izumi S, Moriuchi T, Koji T, Tanno M, Terashima K, Iguchi K, Mochizuki T, Yanaihara C, Yanaihara T, Abe K, Nakane PK (1988) Localization of c-myc in HL-60 cells, neoplastic and normal tissues: An immunohistochemistry and in situ hybridization study. Acta Histochem Cytochem 20: 327–342
Koji T, Sugawara I, Kimura M, Nakane PK (1989) In situ localization of c-myc in HL60 cells using non-radioactive synthetic oligonucleotide probes. Acta Histochem Cytochem 22: 295–307
Nakane PK (to be published) Modern histochemical methods in mucosal immunology. In: Proc Vlth International Congress of Mucosal Immunology, Elsevier, Amsterdam
Yamada G, Takahashi M, Endo H, Takaguchi K, Nishimoto H, Tsuji T (to be published) Pathogenesis and interferon treatment in chronic hepatitis C. In: Proc Vlth International Congress of Mucosal Immunology, Elsevier, Amsterdam
Izumi S, Koji T, Nakane PK (1990) In situ localization of mRNA visualized by backscattered electron imaging. J Histochem Cytochem 38: 1052
Mazurkiewicz JE, Nakane PK (1972) Light and electron microscopic localization of antigens in tissues embedded in polyethene glycol with a peroxidase-Iabeled antibody method. J Histochem Cytochem 20: 967–974
Isenberg KE, Mudd J, Shah V, Merlie JP (1986) Nucleotide sequence of the mouse muscle nicotinic acetylcholine receptor alpha subunit. Nucleic Acids Res 14: 5111
Levine L, Seaman E, Hammerschlag E, Van Vunakis H (1966) Antibodies to photoproducts of deoxyribonucleic acids irradiated with ultraviolet light. Science 153: 1666–1667
Wilson, MB, Nakane PK (1978) Recent developments in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies. In: Knapp W, Holubar K, Wick G (eds) Immunofluorescence and related staining techniques. Elsevier/ North-Holland Biomedical, 215–224
Graham RC, Karnovsky MJ (1966) The early stages of injected horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique. J Histochem Cytochem 14: 291–302
Adams JC (1981) Heavy metal intensification of DAB-based HRP reaction product. J Histochem Cytochem 29: 775
Brigati DJ, Myerson D, Leary JJ, Spalholz B, Travis SZ, Fong CKY, Hsiung GD, Ward DC (1983) Detection of viral genomes in cultured cells and paraffin- embedded tissue sections using biotin-labeled hybridization probes: General method for in situ hybridization. Virology 126: 32–50
Karnovsky MJ (1964) The localization of Cholinesterase activity in rat cardiac muscles by electron microscopy. J Cell Biol 23: 217–232
Langer PR, Waldrop AA, Ward DC (1981) Enzymatic synthesis of biotin-labeled polynucleotides: Novel nucleic acid affinity probes. Proc Natl Acad Sei USA 78: 6633–6637
Tchen P, Fuchs RPP, Sage E, Leng M (1984) Chemically modified nucleic acids as immunodetectable probes in hybridization experiments. Proc Natl Acad Sei USA 81: 3466–3470
Forster AC, Mclnnes JL, Skingle DC, Symons RH (1985) Nonradioactive hybri-dization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin. Nucleic Acids Res 13: 745–759
Landegent JE, Jansen in de Wal N, Baan RA, Hoeijmakers JHJ, 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
Franklin WA, Ming Lo K, Haseltine WA (1982) Alkaline lability of fluorescent photoproducts produced in ultraviolet lightirradiated DNA. J Biol Chem 257: 13535–13543
Campisi J, Gray HE, Pardee AB, Dean M, Sonenshein GE (1984) Cell-cycle control of c-myc but c-ras expression is lost following chemical transformation. Cell 36: 241–247
Hutchison NJ, Langer-Safer PR, Ward DC, Hamkalo BA (1982) In situ hybridization at the electron microscope level: Hybrid detection by autoradiography and colloidal gold. J Cell Biol 95: 609–618
Singer RH, Lawrence JB, Silva F, Langevin GL, Pomeroy M, Billings-Gargliardi S (1989) Strategies for ultrastructural visualization of biotinated probes hybridized to messenger RNA in situ. In: Current topics in microbiology and immunology, vol. 143. Springer, Berlin Heidelberg, pp 55–69
Silva FG, Lawrence JB, Singer RH (1989) Progress toward ultrastructural idenfication of individual mRNAs in thin section: Myosin heavy-chain mRNA in developing myotubes. In: Techniques in immunohistochemistry, vol 4. Academic, New York, pp 147–165
Binder M, Tourmente S, Roth J, Renaud M, Gehring W (1986) In situ hybridization at the electron microscopic level: Localization of transcripts on ultrathin sections of Lowcryl K4M-embedded tissue using biotinated probes and protein A-gold complexes. J Cell Biol 102: 1646–1653
Webster H de F, Lamperth L, Favilla J T, Lemke G, Tesin D, Manuelidis L (1987) Use of a biotinylated probe and in situ hybridization for light and electron microscopic localization of Po mRNA in myelin-forming Schwann cells. Histochemistry 86: 441–444
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1991 Springer-Verlag Tokyo
About this chapter
Cite this chapter
Nakane, P.K., Matsumura, H., Koji, T. (1991). In Situ Hybridization Using T-T Dimerized Non-Radioactive Probes. In: Tabuchi, K. (eds) Biological Aspects of Brain Tumors. Springer, Tokyo. https://doi.org/10.1007/978-4-431-68150-2_5
Download citation
DOI: https://doi.org/10.1007/978-4-431-68150-2_5
Publisher Name: Springer, Tokyo
Print ISBN: 978-4-431-68152-6
Online ISBN: 978-4-431-68150-2
eBook Packages: Springer Book Archive