H2AX phosphorylation at Ser139 (formation of γ-H2AX) is an indicator of double-strand breaks in DNA (DSBs) after the action of different genotoxic stresses, including ionizing radiation, environmental agents, and chemotherapy drugs. The sites of DSBs can be visualized as focal sites of γ-H2AX using antibodies and immunofluorescence microscopy. The microscopy technique is the most sensitive method of DSB detection in individual cells. It is useful for experimental research, radiation biodosimetry, and clinical practice. In this chapter, we provide an immunochemical protocol for γ-H2AX labeling and analysis by confocal microscopy. The advantage of the assay is that it enables the quantitation of γ-H2AX foci in individual cells in different phases of the cell cycle.
Keywords: Apoptosis; Confocal microscope; Double-strand breaks; Immunofluorescence; Mammalian cells; γ-H2AX.