Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells

Susan Kilgas, Anne E. Kiltie, Kristijan Ramadan*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

DNA end resection converts broken ends of double-stranded DNA (dsDNA) to 3′-single-stranded DNA (3′-ssDNA). The extent of resection regulates DNA double-strand break (DSB) repair pathway choice and thereby genomic stability. Here, we characterize an optimized immunofluorescence (IF) microscopy-based protocol for measuring ssDNA in mammalian cells by labeling genomic DNA with 5-bromo-2′-deoxyuridine (BrdU). BrdU foci can be detected under non-denaturing conditions by anti-BrdU antibody, providing an accurate and reliable readout of DNA end resection in most mammalian cell lines. For complete details on the use and execution of this protocol, please refer to Kilgas et al. (2021).

Original languageEnglish
Article number100978
Number of pages15
JournalSTAR Protocols
Volume2
Issue number4
Early online date25 Nov 2021
DOIs
Publication statusE-pub ahead of print - 25 Nov 2021

Keywords

  • Antibody
  • Cell Biology
  • Cell-based Assays
  • Microscopy
  • Molecular Biology

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