Assessing DNA Damage Foci: To Identify Radiation-Induced DNA Damage Foci in Cancer Cell Line Using Immunofluorescence Staining

- Ionizing radiation induces different types of DNA damage, such as single-stranded breaks or SSBs and double-stranded breaks or DSBs. Single strand breaks or SSBs occur when the radiation damages the bases and cleaves the DNA backbone, while double strand breaks, DSBs, develop when two SSBs occur on opposite DNA strands. The first step of DSB formation is the phosphorylation of the H2AX histone, which can be visualized by immunofluorescence staining.

To perform immunofluorescence staining, add primary antibodies to a sample fixed on a cover slip placed in a Petri dish. The antibodies bind to phosphorylated histone protein gamma-H2AX. Incubate the sample and wash it with PBS to remove unbound antibodies. Next, add secondary antibodies carrying fluorophores, which recognize primary antibodies and bind to them. Add the desired amount of DAPI fluorescent stain to counter-stain the nuclei.

Gently mount the cover slip on a glass slide with a mounting medium and observe cells under a fluorescent microscope. The gamma-H2AX foci appear as distinct fluorescent dots in the irradiated cancer cells. In the following protocol, we will perform immunofluorescence staining of irradiated human colo