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