Immunofluorescence-Based Assay for Detection of Nuclear RAD51 Foci as a Marker of Homologous Recombination Repair in...

RAD51 is a central protein in the homologous recombination (HR) pathway and is essential for the accurate repair of DNA double-strand breaks (DSBs). Following DSB formation, DNA end resection generates single-stranded DNA substrates that facilitate the recruitment and assembly of RAD51 nucleoprotein filaments at sites of damage. This process results in the formation of discrete nuclear RAD51 foci, which serve as a widely accepted functional readout of HR activity and a surrogate marker of HR proficiency. Because defects in HR are common in several malignancies, particularly ovarian and breast cancers, assessment of RAD51 foci formation has emerged as an important approach for evaluating DNA repair capacity and predicting response to DNA-damaging therapies, including platinum compounds and poly(ADP-ribose) polymerase (PARP) inhibitors, whose efficacy is strongly influenced by HR repair status. This manuscript describes a simple, reliable, and reproducible immunofluorescence-based protocol for the detection and quantification of RAD51 nuclear foci in cultured ovarian cancer cells. The method involves induction of DNA damage by ionizing radiation (IR), followed by fixation, immunostaining with antibodies against RAD51 and γH2AX, confocal microscopy, and manual quantitative analysis of RAD51/γH2AX co-localized foci. The protocol can be applied under basal conditions or after genetic and pharmacological perturbations to determine their effects on HR function. Representative results demonstrate robust induction of RAD51 foci in HR-proficient ovarian cancer cells following DNA damage, whereas RAD51 depletion markedly reduces foci formation despite comparable levels of DSBs, confirming assay specificity. Overall, this protocol provides a robust and reproducible functional assay for assessing HR competency, with broad applications in preclinical and potentially translational cancer research.