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Journal of Histochemistry and Cytochemistry, Vol. 46, 1203-1210, October 1998, Copyright © 1998, The Histochemical Society, Inc.

TECHNICAL NOTE

A New Immunocytochemical Technique for Ultrastructural Analysis of DNA Replication in Proliferating Cells After Application of Two Halogenated Deoxyuridines

Françoise Jaunina, Astrid E. Visserb, Dusan Cmarkoa, Jacob A. Atenb, and Stanislav Fakana
a Centre of Electron Microscopy, University of Lausanne, Lausanne, Switzerland
b Centre for Microscopical Research, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands

Correspondence to: Stanislav Fakan, Centre of Electron Microscopy, Univ. of Lausanne, 27 Bugnon, CH-1005 Lausanne, Switzerland..

We describe a colloidal gold immunolabeling technique for electron microscopy which allows one to differentially visualize portions of DNA replicated during different periods of S-phase. This was performed by incorporating two halogenated deoxyuridines (IdUrd and CldUrd) into Chinese hamster cells and, after cell processing, by detecting them with selected antibodies. This technique, using in particular appropriate blocking solutions and also Tris buffer with a high salt concentration and 1% Tween-20, prevents nonspecific background and crossreaction of both antibodies. Controls such as digestion with DNase and specific staining of DNA with osmium ammine show that labeling corresponds well to replicated DNA. Different patterns of labeling distribution, reflecting different periods of DNA replication during S-phase, were characterized. Cells in early S-phase display a diffuse pattern of labeling with many spots, whereas cells in late S-phase show labeling confined to larger domains, often at the periphery of the nucleus or associated with the nucleolus. The good correlation between our observations and previous double labeling results in immunofluorescence also proved the technique to be reliable.

(J Histochem Cytochem 46:1203–1209, 1998)

Key Words: deoxyuridines, DNA replication, double labeling, electron microscopy, IdUrd, CldUrd, immunogold labeling, S-phase


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