Staining Against Phospho-H2AX (gamma-H2AX) as a Marker for DNA Damage and Genomic Instability in Cancer Tissues and Cells

Abstract

Phospho-H2AX or gamma-H2AX- is a marker of DNA double-stranded breaks and can therefore be used to monitor DNA repair after, for example, irradiation. In addition, positive staining for phospho-H2AX may indicate genomic instability and telomere dysfunction in tumour cells and tissues. Here, we provide a protocol to perform immunostaining for phospho-H2AX on cells, cryosections and formalin-fixed, paraffin-embedded tissues. Crucial steps in the protocol and troubleshooting suggestions are indicated. We also provide suggestions on how to combine staining against gamma-H2AX with stainings against components of the tumour microenvironment, such as hypoxia and blood vessels.