Radiation-Induced Gamma-H2AX Foci Staining and Analysis.

DNA damage induces activation of DNA damage-associated proteins. In the case of DNA double-strand breaks (DSBs), H2A histone family member X (H2AX) plays a crucial role in DNA repair. Upon DSBs, H2AX is phosphorylated at serine 139, forming γH2AX, which creates characteristic "foci" at the site of damage. These foci can be visualized through fluorescent immunostaining under a microscope, allowing for the counting of individual DSBs within nuclei. This technique is superior to previously established DSB detection assays, such as neutral elution, pulse field gel electrophoresis, and neutral comet assays, in terms of sensitivity for detecting single DSBs. Further research supports the idea that this assay provides valuable insight into the dynamics of DSB responses and repair mechanisms. In this chapter, we present a technique to target and visualize the foci of γH2AX by fluorescent immunocytochemical methods.