An Immunofluorescence-Based Method to Quantify Replication Stress in Cancer Cells

Begin with cancer cells carrying IdU, a synthetic substitute of thymidine nucleotide, already integrated into their DNA. When these cells encounter replication stress, their DNA synthesis halts, revealing IdU within exposed single-stranded DNA regions.

Fix the cells and add detergent molecules to make the cell membrane permeable.

Add BSA to block non-target antibody binding sites.

Introduce anti-IdU antibodies that interact with IdU-labeled single-stranded DNA.

Add green fluorophore-labeled secondary antibodies that target anti-IdU antibodies. Remove the unbound antibodies.

Place the coverslip on a slide containing a mounting medium with a fluorescent dye — that stains the nuclei.

Under a fluorescence microscope, observe the blue nuclei.

The green fluorescence foci represent antibodies binding to the single-stranded DNA.

Count the number of foci to quantify the level of replication stress.