An optimized proximity ligation assay to detect telomere dysfunction induced foci in human and mouse cells

Yajun Wang; Luigi Ferrucci; Michael M Seidman; Yie Liu

doi:10.1016/j.xpro.2022.101610

An optimized proximity ligation assay to detect telomere dysfunction induced foci in human and mouse cells

STAR Protoc. 2022 Aug 12;3(3):101610. doi: 10.1016/j.xpro.2022.101610. eCollection 2022 Sep 16.

Authors

Yajun Wang¹, Luigi Ferrucci², Michael M Seidman³, Yie Liu⁴

Affiliations

¹ Laboratory of Genetics and Genomics, National Institute on Aging, NIH, 251 Bayview Blvd, Baltimore, MD 21224, USA.
² Translational Gerontology Branch, National Institute on Aging, NIH, 251 Bayview Blvd, Baltimore, MD 21224, USA.
³ Laboratory of Molecular Biology and Immunology, National Institute on Aging, NIH, 251 Bayview Blvd, Baltimore, MD 21224, USA.
⁴ Laboratory of Genetics and Genomics, National Institute on Aging, NIH, 251 Bayview Blvd, Baltimore, MD 21224, USA. Electronic address: liuyie@mail.nih.gov.

Abstract

Telomere dysfunction-induced foci (TIF) can be measured by immunofluorescence, combined with telomere-fluorescent in situ hybridization. We modified this approach by combining the proximity ligation assay (PLA), which detects colocalization of two molecules in proximity through a signal amplification step and improves the fidelity and sensitivity of TIF detection in human and mouse cells. The protocol includes cell preparation, permeabilization, fixation, and blocking PLA detection of DNA damage response proteins within proximity with telomeres and optional PLA verification by immunofluorescence-based technique.

Keywords: Antibody; Cell biology; In Situ hybridization; Microscopy; Molecular biology.

Publication types

Research Support, N.I.H., Intramural

MeSH terms

Animals
Fluorescent Antibody Technique
Humans
In Situ Hybridization, Fluorescence
Mice
Telomere*