Encyclopedia of Experiments: Biological Techniques
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Transcript
Radiation-induced DNA double-strand breaks, or DSBs, initiate DNA repair protein recruitment at the damaged site — activating DNA repair.
To detect the DNA repair proteins γH2AX and 53BP1 at DSB sites using indirect immunofluorescence, take coverslips containing epithelial cells. Irradiate a subset of the cells. Incubate, allowing the control non-irradiated and irradiated cells to grow for specific times.
Treat with a buffer to extract the cell nuclei. Fix the cell nuclei to preserve their morphology. Treat with a blocking solution to minimize non-specific primary antibody binding.
Add primary antibodies targeting the two DNA repair proteins. The antibodies bind to the respective proteins.
Incubate with secondary antibodies tagged with red and green fluorophores, respectively. The secondary antibodies — specific for each primary antibody — bind to their targets. Add mounting medium containing DAPI on a glass slide. Mount the coverslips on it for imaging.
DAPI — a DNA-binding dye — binds to the AT-rich minor grooves of the double-stranded DNA, counterstaining the nuclei. Visualize the cell nuclei using a fluorescence microscope.
Compared to non-irradiated cell nuclei, irradiated nuclei exhibit multiple γH2AX foci that decrease over time, indicating progressive DNA repair in cells grown for longer periods.
Overlapping red and green fluorescence signals appear yellow, indicating the co-localization of γH2AX and 53BP1 at the DSB site following irradiation.
