Use of halogenated precursors for simultaneous DNA and RNA detection by means of immunoelectron and immunofluorescence microscopy.
We have developed a novel approach for in situ labeling and detection of nucleic acids in cultured cells. It is based on in vivo incorporation of chlorouridine (ClU) or iododeoxyuridine (IdU) into Chinese hamster ovary cells with the aim of labeling RNA and DNA, respectively. The halogenated nucleotides are immunolabeled on ultrathin sections with anti-bromodeoxyuridine (BrdU) monoclonal antibodies that specifically react with either IdU or ClU. Furthermore, we combined ClU and IdU incubation to label simultaneously RNA and DNA in the same cell. Both were visualized by means of anti-BrdU antibodies exhibiting strong affinity for one of the two halogenated epitopes. Confocal imaging of interphase nuclei and electron microscopic analysis showed evidence of a partial colocalization of newly synthesized DNA and RNA inside the cell nucleus. RNase and DNase digestion of ultrathin sections after formaldehyde fixation and acrylic resin embedding confirmed the specificity of incorporation. Consequently, this method allows us to differentially label DNA and RNA on the same section. Using short pulses with the precursors, we could show that newly synthesized DNA and RNA both preferentially occur within the perichromatin region at the border of condensed chromatin domains.
Vecchio
Lorella
L
Centre of Electron Microscopy, University of Lausanne, Rue du Bugnon 27, CH 1005 Lausanne, Switzerland.
Solimando
Liliana
L
Biggiogera
Marco
M
Fakan
Stanislav
S
D000818:Animals; D000906:Antibodies; D001973:Bromodeoxyuridine / Q000276:immunology; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003429:Cross Reactions; D004247:DNA / Q000032:analysis*; D003857:Deoxyuridine / Q000031:analogs & derivatives*; D007065:Idoxuridine; D008856:Microscopy, Fluorescence / Q000379:methods; D016253:Microscopy, Immunoelectron / Q000379:methods; D012313:RNA / Q000032:analysis*
D000906:Antibodies; C012244:5-chloro-2'-deoxyuridine; D012313:RNA; D004247:DNA; D001973:Bromodeoxyuridine; D007065:Idoxuridine; D003857:Deoxyuridine