Ultrastructural detection of DNA within the nucleolus by sensitive molecular immunocytochemistry.
This paper describes a new technique for locating DNA on semithin or ultrathin sections of aldehyde-fixed and plastic-embedded cells or tissues. Sections were incubated in a medium containing bromodeoxyuridine (BUdR) triphosphate and terminal deoxynucleotidyltransferase. The labeled nucleotides bound at the surface of the sections were subsequently detected with an anti-BUdR antibody and immunoglobulin-gold complex. On semithin sections, labeled nucleotide detection was achieved by an amplification step with silver enhancement. This technique was applied to a wide variety of biological materials allowing a sensitive detection of DNA-containing structures, even where these are present in very low amounts. Examples of high resolution and sensitive detection include the DNA present in mitochondria, chloroplasts, mycoplasmas, and DNA viruses. Special attention focused on the location of DNA inside the nucleolus. In Ehrlich tumor cell nucleoli, DNA was detected in the fibrillar centers and not in the dense fibrillar component. Identical results were found in the nucleoli of other cell types. These results contradict earlier data but conform with other recent immunocytochemical observations concerning the correlation between structure and function in the nucleolus. This method provides a useful tool for investigations requiring highly precise correlations between a molecular function and a given ultrastructural morphology.
Thiry
M
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Laboratory of Cell and Tissue Biology, University of Liège, Belgium.
D000818:Animals; D002466:Cell Nucleolus / Q000737:chemistry* / Q000648:ultrastructure; D002645:Chickens; D002696:Chlamydomonas / Q000737:chemistry; D004247:DNA / Q000032:analysis*; D004253:DNA Nucleotidylexotransferase; D003856:Deoxyuracil Nucleotides; D005347:Fibroblasts / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry; D007583:Jejunum / Q000737:chemistry; D051381:Rats
D003856:Deoxyuracil Nucleotides; C032549:bromodeoxyuridine triphosphate; D004247:DNA; D004253:DNA Nucleotidylexotransferase