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

Abstract

Summary: 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. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell biology
• Microscopy
• Molecular biology
• Antibody
• In Situ hybridization