Summary
For the first time a monoclonal antibody against 5-bromodeoxyuridine was used to detect cell proliferation in pig testis and intestine sections. The influence of several parameters such as mode of injection, addition of thymidine biosynthesis inhibitor, tissue fixation, hydrolysis and revelation was examined. The technique of choice consisted in intravenously injecting the animals with 50 mg/kg BUdR added to 10 mg/kg FUdR 2 h before tissue collection and Bouin fixation; hydrolysis of sections was performed by HCl 4N: Ethanol 70° (1:1 v/v); revelation of BUdR was made by a secondary antibody linked to colloidal gold particles, followed by a silver enhancement step. The data were superior when compared to those obtained by direct immunofluorescence and by the PAP technique. The described method is convenient and sensitive, provides an intense nuclear labelling without background and allows simultaneous examination of histology. The advantages over the technique using tritiated thymidine are particularly obvious when fast screening of numerous samples is required or when new experimental protocols are developping.
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References
Danscher G (1981a) Histochemical detection of heavy metals. A revised version of the sulphide silver method suitable for both light and electron microscopy. Histochemistry 71:1–16
Danscher G (1981b) Light and electron microscopic localisation of silver in biological tissue. Histochemistry 71:177–186
De Mey J, Hacker GW, De Waele M, Springall DR (1986) Gold probes in light microscopy. In: Polak JM, Van Noorden S (eds) Immunohistochemistry — modern methods and applications. 2nd edn. Wright, Bristol, pp 92–110
De Waele M, De Mey J, Renmans W, Labeur C, Jochmans K, Van Camp B (1986) Potential of immunogold-silver staining for the study of leukocyte subpopulations as defined by monoclonal antibody. J Histochem Cytochem 34:1257–1263
Ellwart J, Dörmer P (1985) Effect of 5-fluoro-2-deoxyuridine (FdUrd) on 5-bromo-2-deoxyurindine (BrdUrd) incorporation into DNA measured with a monoclonal BrdUrd antibody and by BrdUrd/Hoechst quenching effect. Cytometry 6:513–520
Gomez F, Pirens G, Schaus C, Closset J, Hennen G (1984) A highly sensitivie radioimmunoassay for human growth hormone using a monoclonal antibody. J Immunoassay 5:145
Gratzner HG (1982) Monoclonal antibody to 5-bromo and 5-iododeoxyuridine: a new reagent for detection of DNA replication. Science 218:474–475
Gunduz N (1985) The use of FITC-conjugated monoclonal antibodies for determination of S-phase cells with fluorescence microscopy. Cytometry 6:597–601
Hacker von GW, Polak JM, Springall DR, Tang SK, Van Noorden S, Lackie P, Grimelius L, Adam H (1985) Immunogold-silver staining. Eine Übersicht. Mikroskopie (Wien) 42:318–325
Harms G, Van Goor H, Koudstaal J, de Ley L, Hardonck MJ (1986) Immunohistochemical demonstration of DNA incorporated 5-bromodeoxyuridine in frozen and plastic embedded sections. Histochemistry 85:139–143
Holgate CS, Jackson P, Cowen PN, Bird CE (1983) Immunogold silver staining: new method with enhanced sensitivity. J Histochem Cytochem 31:938–944
Jackowski G, Suria JD, Liew CC (1976) Fractionation of nucleolar proteins by two-dimensional gel electrophoresis. Can J Biochem 54:9–14
Kornberg A (1980) DNA replication. WH Freeman, San Francisco, 724 pp
Lassoie L (1983) Cours de chirurgie vétérinaire. Administration et soustraction chirurgicales, cathétérisme. Office des cours de la Faculté de Médecine Vétérinaire de l'Université de Liège, 92 pp
Mather JP, Saez JM, Haour F (1981) Primary cultures of Leydig cells from rat, mouse and pig: advantages of porcine cells for the study of gonadotropin regulation of Leydig cell function. Steroids 38:35–44
Morstyn G, Pyke K, Gardner J, Ashcroft F, de Fazio A, Bhathal P (1986) Immunohistochemical identification of proliferating cells in organ culture using bromodeoxyuridine and a monoclonal antibody. J Histochem Cytochem 34:697–701
Muramatsu M, Hayashi Y, Onishi T, Sakai M, Takai K, Kashiyama K (1974) Rapid isolation of nuclei from detergent purified nuclei of various tumor and tissue culture cells. Exp Cell Res 88:345–351
Nagashima T, De Armond SJ, Murocvic J, Hishimo T (1985) Immunocytochemical demonstration of S-phase cells by anti-bromodeoxyuridine monoclonal antibody in human brain tumor tissue. Acta Neuropathol 67:155–159
Perrard-Sapori MH, Saez JM, Dazord A (1985) Hormonal regulation of proteins secreted by cultured pig Sertoli cells: characterization by two-dimensional polyacrylamide gel electrophoresis. Mol Cell Endocrinol 43:188–197
Raza A, Vear K, Preisler HD (1985) Double labeling and in vitro versus in vivo incorporation of bromodeoxyuridine in patients with acute non lymphocytic leukemia. Cytometry 6:633–640
Sternberger L (1979) Immunocytochemistry. 2nd edn. John Wiley and Sons, New York
Sugihara H, Hattori T, Fukuda M (1986) Immunohistochemical detection of bromodeoxyuridine in formalin-fixed tissue. Histochemistry 85:193–195
Vandalem J-L, McNamara M, Colenbrander N, McDonald A, Van de Wiel D, Meijer J, Hennen G (1986) Hemicastration in the normal and the cryptochid pig: effects on gonadotropins and gonadotropin receptors. European Testis Workshop, Rome (Italy). Abstract
Varndell IM, Polak JM, Sikri KL, Minth CD, Bloom SR, Dixon JE (1984) Visualisation of messenger RNA directing peptide synthesis by in situ hybridization using a novel single—stranded cDNA probe. Potential for the investigation of gene expression and endocrine cell activity. Histochemistry 81:597–601
Wynford-Thomas D, Williams ED (1986) Use of bromodeoxyuridine for cell kinetic studies in intact animals. Cell Tissue Kinet 19:179–182
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Dombrowicz, D., Delahaut, P., Danguy, A. et al. Detection of cell proliferation in pig testis and intestine sections using monoclonal anti-bromodeoxyuridine antibody and immunogold silver staining. Histochemistry 90, 31–35 (1988). https://doi.org/10.1007/BF00495703
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DOI: https://doi.org/10.1007/BF00495703