Improved identification of DNA double strand breaks: γ-H2AX-epitope visualization by confocal microscopy and 3D reconstructed images

Radiation and Environmental Biophysics
Nico RuprechtJohannes T Heverhagen

Abstract

Currently, in the context of radiology, irradiation-induced and other genotoxic effects are determined by visualizing DSB-induced DNA repair through γ-H2AX immunofluorescence and direct counting of the foci by epifluorescence microscopy. This procedure, however, neglects the 3D nature of the nucleus. The aim of our study was to use confocal microscopy and 3D reconstructed images to improve documentation and analysis of γ-H2AX fluorescence signals after diagnostic examinations. Confluent, non-dividing MRC-5 lung fibroblasts were irradiated in vitro with a Cs-137 source and exposed to radiation doses up to 1000 mGy before fixation and staining with an antibody recognizing the phosphorylated histone variant γ-H2AX. The 3D distribution of γ-H2AX foci was visualized using confocal laser scanning microscopy. 3D reconstruction of the optical slices and γ-H2AX foci counting were performed using Imaris Image Analysis software. In parallel, γ-H2AX foci were counted visually by epifluorescence microscopy. In addition, whole blood was exposed ex vivo to the radiation doses from 200 to 1600 mGy. White blood cells (WBCs) were isolated and stained for γ-H2AX. In fibroblasts, epifluorescence microscopy alone visualized the entirety of fluoresc...Continue Reading

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