Superiority of in situ hybridization over immunolabeling for detecting DNA on Lowicryl sections: a study on adenovirus-infected cells.
We investigated the intranuclear distribution of viral genomes in adenovirus Type 5-infected HeLa cells on ultra-thin sections of Lowicryl K4M-embedded material by immunolabeling of DNA and in situ hybridization with a viral DNA probe. Monoclonal AC-30-10, raised against double- and single-stranded DNA (dsDNA, ssDNA), and HB2 antibodies, raised against only the dsDNA, were used. Both antibodies intensely labeled the condensed host chromatin and the virus-induced substructures containing inactive encapsidated and non-encapsidated viral genomes. The antibodies labeled only slightly those substructures containing replicating and transcribing viral genomes. Viral ssDNA, which is accumulated within well-delineated compact fibrillar structures, was not revealed by AC-30-10 even after elimination of the proteins of the section. In situ hybridization detected all of these types of viral DNA, depending on the protocol used. Therefore, the sensitivity of in situ hybridization for detecting DNA appears superior to immunolabeling with specific antibodies.
Puvion-Dutilleul
F
F
Laboratoire de Biologie et Ultrastructure du Noyau, UPR272 CNRS, Villejuif, France.
Pichard
E
E
D000256:Adenoviridae / Q000235:genetics*; D000911:Antibodies, Monoclonal; D002843:Chromatin / Q000737:chemistry; D015342:DNA Probes; D004277:DNA, Single-Stranded / Q000032:analysis; D004279:DNA, Viral / Q000032:analysis*; D010450:Endopeptidases / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells / Q000382:microbiology; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization
D000911:Antibodies, Monoclonal; D002843:Chromatin; D015342:DNA Probes; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D010450:Endopeptidases