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 CHO cells with the aim of labeling RNA and DNA, respectively. The halogenated nucleotides are then immunolabeled on ultrathin sections with anti-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 as well as electron microscopic analysis put in evidence 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 pulse 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.
The use of halogenated precursors for simultaneous DNA and RNA detection by means of immunoelectron and immunofluorescence microscopy.
Biggiogera M;
2008
Abstract
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 CHO cells with the aim of labeling RNA and DNA, respectively. The halogenated nucleotides are then immunolabeled on ultrathin sections with anti-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 as well as electron microscopic analysis put in evidence 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 pulse 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
