Dapi 4 6 diamidino 2 phenylindole

A DSB marker, γ-H2AX foci formation assay was carried out as it was previously (23 (link)). Briefly, each slide containing cells was taken out of the flask following irradiation and was washed in cold PBS and fixed for 15 min in 4% w/v paraformaldehyde in PBS and washed again in PBS. Cells were then permeabilized for 5 min in 0.2% v/v Triton X-100 (Sigma, St Louis MO, USA) in PBS and washed twice in PBS. Slides were treated with 10% goat serum for 1 h at 37°C for blocking. Antibodies were diluted with 10% v/v goat serum in PBS. Cells were incubated with 1:300 diluted mouse anti-γ-H2AX antibody (Millipore, Burlington, MA, USA) for 1 h at 37°C, washed three times in PBS, and incubated with 1:500 diluted Alexa Fluor 488 goat anti-mouse IgG antibody (Abcam, Cambridge, MA, USA) for 1 h at 37°C, and washed four times in PBS. DAPI (4′,6-diamidino-2-phenylindole) (Roche, Indianapolis, IN, USA) in SlowFade was then applied to stain the DNA.

Prior to liver harvesting by snap-freezing (TissueTek, Sakura, Japan) mice were perfused intraarterially with saline 0.9% upon euthanasia to minimize blood cell autofluorescence. Cryosections (5 μm) were fixed in 4% paraformaldehyde for 15 min, blocked with 10% donkey serum in PBS, and subsequently stained with rabbit anti-HA antibody (1:100), donkey anti-rabbit-cy3 (1:200, Dianova, Hamburg, Germany), and DAPI (4′,6-diamidino-2-phenylindole, 1:10000, Roche, Mannheim, Germany). GFP fluorescence was preserved during this procedure. A BZ-9000 microscope (Keyence, Japan) was used for visualization.

Cells were seeded onto 24 × 24 mm glass coverslips and incubated under the indicated growth conditions until they were fixed for 15 min using 3.7% paraformaldehyde in phosphate-buffered saline (PBS), followed by 0.1% Triton X-100 for permeabilization for 5 min, and then blocked in blocking solution (PBS with 10% FCS and 5% bovine serum albumin, Roche Applied Science, Penzberg, Germany) for 2 h at room temperature. Cells were incubated with primary antibodies from different species overnight at 4 °C under a humidified atmosphere. Immunostained coverslips were then incubated with the corresponding secondary antibody for 1 h at room temperature, counterstained by DAPI (4′,6-diamidino-2-phenylindole, Roche Diagnostics Gmbh, Mannheim, Germany) to visualize nuclei, and mounted on glass slides with Mowiol (Sigma, St. Louis, MI, USA)-based mounting medium. Coverslips were rinsed in PBS between each step. Slides were later analyzed with an Olympus invert microscope IX71 equipped with a CCD camera (CAM-XM10) using Cell^M/Cell^R software (version 3.2) (Olympus, Shinjuku, Japan) or a confocal laser scan microscope (Leica SP5, Leica, Mannheim, Germany) using Leica confocal LAS AF software (version 3.3.10134). During the analysis of subcellular distribution, cells showing apoptosis or extremely high expression levels were avoided.