Abstract
A new method of the light microscopy detection of BrdU-labeled DNA in situ is described. It is based on the oxidative attack at the deoxyribose moiety by copper(I) in the presence of oxygen, which leads to the abstraction of hydrogen atom from deoxyribose culminating in the elimination of the nucleobase, scission of the nucleic-acid strand and formation of frequent gaps. The gaps allow the reaction of the antibodies with the commonly used markers of replication (e.g. 5-bromo-2'-deoxyuridine), which are otherwise masked. The method developed makes it possible to detect nuclear and mitochondrial DNA replication efficiently. In most cases, it does not inhibit effective protein detections and in addition enables simultaneous localization of newly-synthesized RNA. The alternative presently-used methods result in protein denaturation and/or extensive DNA cleavage followed by the DNA-bound proteins peeling off.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Ascorbic Acid / chemistry
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Bromodeoxyuridine / chemistry*
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Bromodeoxyuridine / metabolism
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Cell Nucleus / metabolism
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Copper Sulfate / chemistry
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DNA Cleavage*
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DNA Replication*
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DNA, Mitochondrial / chemistry
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DNA, Mitochondrial / genetics
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Deoxyribonuclease I
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Fluorescent Antibody Technique, Indirect
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HeLa Cells
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Humans
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Oxidation-Reduction
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Oxygen / chemistry
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Staining and Labeling
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Superoxide Dismutase / chemistry
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Superoxides / chemistry
Substances
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DNA, Mitochondrial
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Superoxides
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Superoxide Dismutase
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Deoxyribonuclease I
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Bromodeoxyuridine
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Copper Sulfate
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Ascorbic Acid
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Oxygen
Grants and funding
This work has been supported by the Czech Science Foundation [204/09/0973, P302/12/G157] and the Grant Agency of the Academy of Sciences of the Czech Republic [KAN 200520801, AV0Z50520514 and AV0Z50040702]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.