Staining Against Phospho-H2AX (γ-H2AX) as a Marker for DNA Damage and Genomic Instability in Cancer Tissues and Cells.
Phospho-H2AX or γ-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 γ-H2AX with stainings against components of the tumour microenvironment, such as hypoxia and blood vessels.
Department of Molecular Materials, Institute for Molecules and Materials, Radboud University, Nijmegen, The Netherlands.
Anika
A
Nagelkerke
Department of Radiation Oncology, RadboudUMC, Nijmegen, The Netherlands. Paul.Span@Radboudumc.nl.
Paul N
PN
Span
D015415:Biomarkers / Q000378:metabolism; D016616:Cryoultramicrotomy; D004249:DNA Damage; D042822:Genomic Instability; D006657:Histones / Q000378:metabolism*; D006801:Humans; D009369:Neoplasms / Q000235:genetics*; D016612:Paraffin Embedding; D010766:Phosphorylation; D013194:Staining and Labeling / Q000379:methods*; D016707:Tissue Fixation
D015415:Biomarkers; C468783:H2AX protein, human; D006657:Histones
DNA damage repair; Genomic instability; Histone 2A; Immunocytochemistry; Immunofluorescence; Immunohistochemistry; Telomere dysfunction