Anti fade mounting solution

The immunofluorescence staining was described previously[6 (link), 8 (link), 15 (link)]. Briefly, harvested BMDCs were seeded in 8-well chamber slides at 2×10⁵ cells/chamber and cultured with 10% FBS RPMI-1640 medium overnight. Cells were starved for 2 hours in pre-warmed SFM and then treated with CpG-B (1μM) for 30 minute or left untreated. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with 5% BSA in PBS, and stained with primary anti-DNA-PKcs and anti-TRAF3 Abs in PBS with 2% BSA at 4°C overnight. Alexa fluorescence-conjugated secondary Abs (Alexa 488 or 594) (Lifetechnologies, NY, USA) was added and incubated for 2 hours. Slides were mounted with anti-fade mounting solution (Electron Microscopy Sciences, PA, USA) and observed under an IX81 Olympus microscope with 60X oil immersion objective powered by 1.6x magnification. The images were captured by an ORCA R2 CCD mono camera and processed by the Metamorph Image Analysis Software (Olympus, Japan).

The frozen tissue sections were fixed with 4% paraformaldehyde for 10 min, washed with PBS-T, and blocked in 3% BSA-PBS for 30 min. The primary antibodies, anti-γ-H2AX (catalog no. PA5-77995; Invitrogen; 1:100) and CD31 (BD Bioscience; clone MEC 13.3; 1:100), were added to the tissue sections and incubated overnight at 4°C. The sections were washed with PBS-T and incubated with the indicated fluorochrome-conjugated secondary antibodies (Molecular Probes) for 1 h at room temperature in the dark. The nuclei were counterstained with DAPI (4′,6- diamidino-2-phenylindole), and the slides were mounted in an antifade mounting solution (Electron Microscopy Sciences). Immunofluorescent images were acquired using a Leica DMI6000B/DFC340 FX fluorescence microscope system. For image acquisition, 1,600- by 1,200-pixel full-frame pictures of various channels were recorded as 16-bit TIFF files with ×40 or ×100 magnification. To quantify the fluorescence signal (Fig. 5), we calculated the corrected total cell fluorescence (CTCF) using ImageJ. The formula for CTCF calculation was CTCF = integrated density − (area of total cell × mean fluorescence of background). For each group, a minimum of 20 images from one experiment were taken, and the results of two independent experiments were plotted as a bar graph.