Quantitation of γH2AX Foci in Tissue Samples

Abstract

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming gammaH2AX, is an early response to DNA double-strand breaks. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR). Overall, DSB induction results in the formation of discrete nuclear gammaH2AX foci which can be easily detected and quantitated by immunofluorescence microscopy. Given the unique specificity and sensitivity of this marker, analysis of gammaH2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of gammaH2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, gammaH2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of gammaH2AX foci ex vivo and in vivo. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of gammaH2AX foci in mouse tissues.