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

• Published in: STAR protocols Vol. 3; no. 3; p. 101610
• Main Authors: Wang, Yajun, Ferrucci, Luigi, Seidman, Michael M., Liu, Yie
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 16.09.2022; Elsevier
• Subjects: Antibody; Cell biology; In Situ hybridization; Microscopy; Molecular biology; Protocol; In Situ hybridization; Cell biology; Microscopy; Molecular biology; Antibody
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2022.101610

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. [Display omitted] •PLA detecting DNA damage response proteins within proximity with telomeres•Optional PLA verification by immunofluorescence-based technique•Increases the fidelity and sensitivity of TIF detection in human and mouse cells•Suitable for detection of telomere dysfunction at single-cell level Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.