biostudies-literatureSmolka JANational Institutes of Healthe202004079https://www.ebi.ac.uk/biostudies/studies/S-EPMC8040515biostudies-literatureUnknown220(6)The S9.6 antibody is broadly used to detect RNA:DNA hybrids but has significant affinity for double-stranded RNA. The impact of this off-target RNA binding activity has not been thoroughly investigated, especially in the context of immunofluorescence microscopy. We report that S9.6 immunofluorescence signal observed in fixed human cells arises predominantly from ribosomal RNA, not RNA:DNA hybrids. S9.6 staining was unchanged by pretreatment with the RNA:DNA hybrid-specific nuclease RNase H1, despite verification in situ that S9.6 recognized RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to RNase T1, which specifically degrades RNA. Additional imaging and biochemical data indicate that the prominent cytoplasmic and nucleolar S9.6 signal priThe Journal of cell biologyRecognition of RNA by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids.PMC8040515T32 GM007377R01 GM120607Hartono SRSmolka JASanz LAChedin FfalseRecognition of RNA by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids.The S9.6 antibody is broadly used to detect RNA:DNA hybrids but has significant affinity for double-stranded RNA. The impact of this off-target RNA binding activity has not been thoroughly investigated, especially in the context of immunofluorescence microscopy. We report that S9.6 immunofluorescence signal observed in fixed human cells arises predominantly from ribosomal RNA, not RNA:DNA hybrids. S9.6 staining was unchanged by pretreatment with the RNA:DNA hybrid-specific nuclease RNase H1, despite verification in situ that S9.6 recognized RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to RNase T1, which specifically degrades RNA. Additional imaging and biochemical data indicate that the prominent cytoplasmic and nucleolar S9.6 signal pri2021-01-01T00:00:00Z2021 Jun2025-04-21T18:15:30.236Z2022-02-11T13:25:15.622ZS-EPMC80405153383017010.1083/jcb.202004079