Plan apochromat 63 na 1.40 oil immersion objective

For immunofluorescence, spleens were embedded and frozen in OCT, sectioned at 15 μm and mounted on SuperFrostPlus Adhesion glass (Thermo Fisher Scientific). Sections were dehydrated using silica beads, fixed with 4% paraformaldehyde for 10 min and washed with PBS. Samples were blocked using 5% normal goat serum for 2 h before staining. Samples were incubated with antibodies against B220 (RA3-6B2, eBioscience), CD3 (17A2, eBioscience) and F4/80 (BM8, Biolegend) diluted in 5% NGS for 2 h at room temperature in the dark. After staining, samples were washed with PBS at least three times. Samples were then mounted using ProLong Gold Antifade Mountant (Invitrogen) and imaged using an inverted LSM780 microscope (Carl Zeiss) and a plan apochromat 63× NA 1.40 oil-immersion objective (Carl Zeiss). For haematoxylin and eosin (H&E) staining, organs were collected and fixed in 10% formalin. Fixed samples were embedded in paraffin and sectioned at 10 μm, mounted on SuperFrostPlus Adhesion glass and stained using H&E. Mounted samples were imaged using a Nikon SMZ1270 Stereo Microscope. Imaging data were analysed using Fiji (ImageJ) software (NIH).

U2OS cells were transfected with DsRed-PCNA and GFP-TFII-I or GFP-Rev1 along with the appropriate siRNAs. DSBs were introduced in the nuclei of cultured cells by microirradiation with a pulsed nitrogen laser (Spectra-Physics; 365 nm, 10 Hz pulse) [48] (link). The laser system was directly coupled (Micropoint Ablation Laser System; Photonic Instruments, Inc.) to the epifluorescence path of an Axiovert 200 M microscope (Carl Zeiss MicroImaging, Inc.) for immunostaining imaging or time-lapse imaging and focused through a Plan-Apochromat 63×/NA 1.40 oil immersion objective (Carl Zeiss MicroImaging, Inc.). The output of the laser power was set at 60% of the maximum. Time-lapse images were taken with an AxioCam HRm (Carl Zeiss MicroImaging, Inc.). During microirradiation, imaging, or analysis, the cells were maintained at 37°C in 35-mm glass-bottom culture dishes (MatTek Cultureware). The growth medium was replaced by CO₂-independent medium (Invitrogen) before analysis. The images were further processed by ImageJ and Photoshop.

Live cell imaging combined with laser micro-irradiation was performed as previously described (6 (link),7 (link)). Fluorescence was monitored via an Axiovert 200 M microscope (Carl Zeiss, Inc.), with a Plan-Apochromat 63×/NA 1.40 oil immersion objective (Carl Zeiss, Inc.). A 365-nm pulsed nitrogen laser (Spectra-Physics) was directly coupled to the epifluorescence path of the microscope and used to generate DSBs in a defined area of the nucleus. For quantitative analyses, the same amount of DNA damage was generated under standardized micro-irradiation conditions (minimal laser output of 75% for five pulses) in each experiment. Time-lapse images were taken with an AxioCamHRm camera. The fluorescence intensities of micro-irradiated and non-irradiated areas within the cell nucleus were determined using the AxioVision Software, version 4.8 (Carl Zeiss, Inc.). Each data point is the average of ≥10 independent measurements.

Microirradiation with a pulsed 365 nm nitrogen laser (Spectra-Physics; 365 nm, 10 Hz pulse) was used to induce DSBs in the nuclei of U2OS cells. The laser system was directly coupled (Micropoint Ablation Laser System; Photonic Instruments, Inc.) to the epifluorescence path of the microscope (Axiovert 200M (Carl Zeiss MicroImaging, Inc.) for time-lapse imaging and focused through a Plan-Apochromat × 63/NA 1.40 oil immersion objective (Carl Zeiss MicroImaging, Inc.). Laser output was set at 75% of the maximum power, equivalent to the minimal dose required to induce detectable accumulation of YFP-DNA-PKcs in living cells. Time-lapse images were acquired with an AxioCam HRm (Carl Zeiss MicroImaging, Inc.). DNA-PKcs and Ku80 kinetics were calculated as previously described52 (link): fluorescence value of an undamaged spot in the same nuclei was subtracted from the fluorescence intensity of the laser-irradiated spot for every cell at each time point in order to eliminate the fluorescence background of the nucleus. Relative fluorescence intensity at each time point (RF(t)) was calculated as RF(t)=(INt−INpreIR)/(INmax−INpreIR), where IN, fluorescence intensity; INpreIR, IN of the micro-irradiated area before laser damage; INmax, maximum IN in the micro-irradiated area.