Cy3 fluorescently labeled secondary antibody

Next, 1 × 10⁴ cells were seeded in a 24‐well plate containing slides. After different transfection treatments, medium was placed into a constant temperature oven at 37°C with 5% CO₂. Cells were exposed to 8‐Gy radiation. After 24 hours, medium was washed three times with pre‐warmed 1× PBS for 10 minutes each. Air‐dried slides were fixed with 4% formaldehyde in TBS for 15‐20 minutes at room temperature and washed three times with 1× PBS for 10 minutes each. Cells were then permeabilized with 0.2% Triton X‐100 for 4 minutes and washed three times with 1× PBS for 10 minutes each. These cells were then blocked in 5% BSA at room temperature for 30 minutes. γ‐H2AX primary antibody (Abcam) was added at 4°C overnight. Cells were washed three times with 1× PBS for 10 minutes each. Next, Cy3 fluorescently labeled secondary antibody (Abcam) was added for 1 hour at room temperature in darkness. Cells were then washed as before. After soaking in Hoechst's solvent for 15 minutes, the plate was sealed with 95% glycerol. Finally, cells were observed under a fluorescence microscope for photographing, at randomly selected multiple fields of view (800) for the counting of foci. Each experiment was repeated three times.

CMFDA-labeled hBMSCs (100 μl of 1 × 10⁶ cells per mL) were inoculated on either Hep-DAT or VEGF–Hep-DAT scaffolds for 2 days, followed by incubation with serum-free culture medium for 21 days. DATs were then embedded in Leica JUNG tissue-freezing medium. Embedded tissue were cut into 8 μm sections with the Leicacm1850 frozen slicer (Leicacm, Berlin, GER). Sections were then blocked in a 1% BSA solution at room temperature for 1 hour, followed by overnight incubation with primary antibody against Von Willebrand Factor (VWF) (anti-VWF, Abcam, Cambridge, USA). Sections were then rinsed three times with PBS, incubated with Cy3 fluorescently labeled secondary antibody (Abcam), for 1 hour at room temperature, washed again in PBS, and finally stained with DAPI and sealed with anti-quencher sealer. The staining intensity of the samples was calculated and classified as weakly expressed (less than 50% of the cells showing strong staining) or strongly expressed (at least 50% of the cells showing strong staining). To avoid possible biases caused by the selection of the fields to be examined, the samples were evaluated by three independent blind-observers.