Summary
We describe a protocol for the ultrastructural detection of DNA-incorporated bromodeoxyuridine (BUdR) in resin embedded tissue by means of post-embedding immunogold labeling. The paraventricular zone of rat embryos brains was dissected, fixed either in paraformaldehyde or glutaraldehyde, and embedded in LR White. BUdR gold labeling was only found when thin sections were pretreated with 4 N HCl. Other DNA denaturing agents, such as Na ethoxide, formamide, formic acid, heat or HCl at lower concentrations were ineffective. Very little difference in the degree of labeling was found depending on the fixation. This method can be applied to investigate the fine structure of replicating cells in other in vivo conditions, such as human tumors.
Similar content being viewed by others
References
Apte SS, Puddle B (1990) Bromodeoxyuridine (BrdUrd) immunohistochemistry in undecalcified plastic-embedded tissue. Elimination of the DNA denaturation step. Histochemistry 93:631–635
Gratzner HG (1982) Monoclonal antibody to 5-bromo and 5-iododeoxyuridine: a new reagent for detection of DNA replication. Science 218:474–475
Harms G, Goor H van, Koudstaal J, Ley L de, Hardonk MJ (1986) Immunohistochemical demonstration of DNA-incorporated 5-bromodeoxyuridine in frozen and plastic embedded sections. Histochemistry 85:139–143
Hayashi Y, Koike M, Matsutani M, Hoshino T (1988) Effects of fixation time and enzymatic digestion on immunohistochemical demonstration of bromodeoxyuridine in formalin-fixed, paraffin-embedded tissue. J Histochem Cytochem 36:511–514
Hoshino T, Prados M, Wilson CB, Cho Kg, Lee K-S, Davis RL (1989) Prognostic implications of the bromodeoxyuridine labeling index of human gliomas. J Neurosurg 71:335–341
Magaud J-P, Sargent I, Clarke PJ, Ffrench M, Rimokh R, Mason DY (1989) Double immunocytochemical labeling of cell and tissue samples with monoclonal anti-bromodeoxyuridine. J Histochem Cytochem 37:1517–1527
Mazzotti G, Rizzoli R, Galanzi A, Papa S, Vitale M, Falconi M, Neri LM, Zini N, Maraldi NM (1990) High-resolution detection of newly synthesized DNA by anti-bromodeoxyuridine antibodies identifies specific chromatin domains. J Histochem Cytochem 38:13–22
Meyer JS, Nauert J, Koehm S, Hughes J (1989) Cell kinetics of human tumors by in vitro bromodeoxyuridine labeling. J Histochem Cytochem 37:1449–1454
Moran R, Darzynkiewicz Z, Staiano-Coico L, Melamed MR (1985) Detection of 5-bromodeoxyuridine (BrdUrd) incorporation by monoclonal antibodies: role of the DNA denaturation step. J Histochem Cytochem 33:821–827
Newman GR, Jasani B, Williams ED (1983) A simple post-embedding system for the rapid demonstration of tissue antigens under the electron microscope. Histochem J 15:543–555
Schutte B, Reynders MMJ, Bosman FT, Blijham GH (1987) Effect of tissue fixation on anti-bromodeoxyuridine immunohistochemistry. J Histochem Cytochem 35:1343–1345
Thiry M, Dombrowicz D (1988) Anti-bromodeoxyuridine monoclonal antibody: an alternative tool for the identification of replicated DNA at the electron microscope level. Biol Cell 62:99–102
Vogel W, Mehnert K, Pentz S (1990) Demonstration of chromosome replication by BrdU antibody technique and electron microscopy. Hum Genet 84:237–240
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Migheli, A., Mocellini, C. & Giordana, M.T. Ultrastructural detection of DNA-incorporated bromodeoxyuridine in resin embedded brain tissue. Histochemistry 95, 491–494 (1991). https://doi.org/10.1007/BF00315745
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00315745