TECHNICAL NOTE

Rapid Synthesis of Biotin-, Digoxigenin-, Trinitrophenyl-, and Fluorochrome-labeled Tyramides and Their Application for In Situ Hybridization Using CARD Amplification

Correspondence to: Anton H.N. Hopman, Dept. of Molecular Cell Biology and Genetics, University Maastricht, PO Box 616, 6200 MD Maastricht, The Netherlands.

A one-step procedure for the synthesis of different tyramide conjugates, which can be utilized in the catalyzed reporter deposition (CARD) amplification system, is described. Succinimidyl esters of biotin, digoxigenin, and of the fluorochromes fluorescein, rhodamine, aminomethylcoumarine acetic acid, and Cy3 were coupled to tyramine in dimethylformamide (DMF) adjusted to a pH of 7.0–8.0 with triethylamine (TEA). The coupling reaction can be performed within 2 hr and the reaction mixture can be applied without further purification steps. Furthermore, trinitrophenyl (TNP)-tyramide was prepared by adding 2,4,6,-trinitrobenzenesulfonic acid to tyramine dissolved in either MilliQ/DMF basified with TEA or in an NaHCO₃ (pH 9.5) buffer. A subsequent precipitation of the TNP–tyramide resulted in a high-yield isolation of this conjugate. The synthesized tyramide conjugates were applied successfully in single- and multiple-target in situ hybridization (ISH) procedures to detect both repetitive and single-copy DNA target sequences in cell preparations with high efficiency. The described approach provides an easy and fast method to prepare a variety of tyramide conjugates in bulk amounts at relatively low cost.

(J Histochem Cytochem 46:771–777, 1998)

Key Words: (F)ISH, horseradish peroxidase, interphase cytogenetics, fluorescent labeling, enzyme cytochemistry, tyramide conjugates, DNA probes

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J. Pan, H. Yeger, and E. Cutz Innervation of Pulmonary Neuroendocrine Cells and Neuroepithelial Bodies in Developing Rabbit Lung J. Histochem. Cytochem., March 1, 2004; 52(3): 379 - 390. [Abstract] [Full Text] [PDF]

				M. Niku, L. Ilmonen, T. Pessa-Morikawa, and A. Iivanainen Limited Contribution of Circulating Cells to the Development and Maintenance of Nonhematopoietic Bovine Tissues Stem Cells, January 1, 2004; 22(1): 12 - 20. [Abstract] [Full Text] [PDF]

				J. Pan, H. Yeger, and E. Cutz Neuronal Developmental Marker FORSE-1 Identifies a Putative Progenitor of the Pulmonary Neuroendocrine Cell Lineage During Lung Development J. Histochem. Cytochem., December 1, 2002; 50(12): 1567 - 1578. [Abstract] [Full Text] [PDF]

				A. Pernthaler, J. Pernthaler, M. Schattenhofer, and R. Amann Identification of DNA-Synthesizing Bacterial Cells in Coastal North Sea Plankton Appl. Envir. Microbiol., November 1, 2002; 68(11): 5728 - 5736. [Abstract] [Full Text] [PDF]

				A. H. N. Hopman, M. A. F. Kamps, E. J. M. Speel, R. F. M. Schapers, G. Sauter, and F. C. S. Ramaekers Identification of Chromosome 9 Alterations and p53 Accumulation in Isolated Carcinoma in Situ of the Urinary Bladder versus Carcinoma in Situ Associated with Carcinoma Am. J. Pathol., October 1, 2002; 161(4): 1119 - 1125. [Abstract] [Full Text] [PDF]

				A. Pernthaler, J. Pernthaler, and R. Amann Fluorescence In Situ Hybridization and Catalyzed Reporter Deposition for the Identification of Marine Bacteria Appl. Envir. Microbiol., June 1, 2002; 68(6): 3094 - 3101. [Abstract] [Full Text] [PDF]

				I. B. BUCHWALOW, T. PODZUWEIT, W. BOCKER, V. E. SAMOILOVA, S. THOMAS, M. WELLNER, H. A. BABA, H. ROBENEK, J. SCHNEKENBURGER, and M. M. LERCH Vascular smooth muscle and nitric oxide synthase FASEB J, April 1, 2002; 16(6): 500 - 508. [Abstract] [Full Text] [PDF]

				H. Yeger, J. Pan, X. W. Fu, C. Bear, and E. Cutz Expression of CFTR and Cl{-} conductances in cells of pulmonary neuroepithelial bodies Am J Physiol Lung Cell Mol Physiol, September 1, 2001; 281(3): L713 - L721. [Abstract] [Full Text] [PDF]

				A. F. M. Moorman, A. C. Houweling, P. A. J. de Boer, and V. M. Christoffels Sensitive Nonradioactive Detection of mRNA in Tissue Sections: Novel Application of the Whole-mount In Situ Hybridization Protocol J. Histochem. Cytochem., January 1, 2001; 49(1): 1 - 8. [Abstract] [Full Text]

				M. Mueller, K. Wacker, W. F. Hickey, E. B. Ringelstein, and R. Kiefer Co-Localization of Multiple Antigens and Specific DNA : A Novel Method Using Methyl Methacrylate-Embedded Semithin Serial Sections and Catalyzed Reporter Deposition Am. J. Pathol., December 1, 2000; 157(6): 1829 - 1838. [Abstract] [Full Text] [PDF]

				F. M. Raaphorst, F. J. van Kemenade, T. Blokzijl, E. Fieret, K. M. Hamer, D. P. E. Satijn, A. P. Otte, and C. J. L. M. Meijer Coexpression of BMI-1 and EZH2 Polycomb Group Genes in Reed-Sternberg Cells of Hodgkin's Disease Am. J. Pathol., September 1, 2000; 157(3): 709 - 715. [Abstract] [Full Text] [PDF]

				H. Knaut, F. Pelegri, K. Bohmann, H. Schwarz, and C. Nusslein-Volhard Zebrafish vasa RNA but Not Its Protein Is a Component of the Germ Plasm and Segregates Asymmetrically before Germline Specification J. Cell Biol., May 15, 2000; 149(4): 875 - 888. [Abstract] [Full Text] [PDF]

				H. M.J. Kerstens, J. C.M. Robben, P. J. Poddighe, W. J.G. Melchers, H. Boonstra, P. C.M. de Wilde, M. V.E. Macville, and A. G.J.M. Hanselaar AgarCyto: A Novel Cell-processing Method for Multiple Molecular Diagnostic Analyses of the Uterine Cervix J. Histochem. Cytochem., May 1, 2000; 48(5): 709 - 718. [Abstract] [Full Text]

				G. Mayer, R. D. Leone, J. F. Hainfeld, and M. Bendayan Introduction of a Novel HRP Substrate-Nanogold Probe for Signal Amplification in Immunocytochemistry J. Histochem. Cytochem., April 1, 2000; 48(4): 461 - 470. [Abstract] [Full Text]

				G. Mayer and M. Bendayan Immunogold Signal Amplification: Application of the CARD Approach to Electron Microscopy J. Histochem. Cytochem., April 1, 1999; 47(4): 421 - 430. [Abstract] [Full Text]

				E. J.M. Speel, A. H.N. Hopman, and P. Komminoth Amplification Methods to Increase the Sensitivity of In Situ Hybridization: Play CARD(S) J. Histochem. Cytochem., March 1, 1999; 47(3): 281 - 288. [Abstract] [Full Text]

				L. Davidson and R. Keller Neural tube closure in Xenopus laevis involves medial migration, directed protrusive activity, cell intercalation and convergent extension Development, January 10, 1999; 126(20): 4547 - 4556. [Abstract] [PDF]

Guidelines | Subscriptions | About | exPRESS - Current - Archive | Business Information | Contact

The Journal of Histochemistry & Cytochemistry is owned, published, and licensed by The Histochemical Society © 1998