Fig. 4.

DNA repair machines at super-resolution. (A) STED 3D image of Rad50 and Mre11 colocalization in Hela cells. 7.5Gy of X-ray irradiation was delivered to cells growing on coverslips using Radsource RS-2000, and cells were allowed to recover for 8h before fixation. Endogenous Rad50 and Mre11 were detected by immunofluorescence with complementary mouse Atto488 (Mre11) and rabbit Atto549 (Rad50) secondary antibodies. Dotted circles represent approximate nuclear membrane boundaries. Insets show zoomed rectangular boxes. Images were captured at 100 × with oil immersion using a Leica TCS SP8 STED 3× microscope capable of continuous-wave stimulated emission depletion (cwSTED) and gated STED imaging, equipped with a 405 nm diode laser and a tuneable super-continuum White Light Laser (WLL, 470–670 nm) for excitation, as well as a 592 nm continuous-wave depletion laser. (B) Super-resolution image of γH2AX foci in Hela cells. Cells were X-ray irradiated, fixed as above and stained with rabbit antiphospho serine 139 specific H2AX antibody followed by antirabbit Cy3B. Image was captured using a Vutara352 microscope. (C) STED imaging of PCNA using the experimental conditions in (A) with anti-PCNA rabbit primary and rabbit Atto594 secondary antibodies.