Laser scanning confocal microscopy

MSCs were seeded in a 12-well plate and accepted irradiation with 12 Gy X-rays. After 48 h, cells were fixed in 4% PFA in PBS for 20 min at room temperature and washed with PBS. Cells were incubated in PBS containing 3% BSA and 0.1% Triton X-100 for 2 h at room temperature. Cells were then incubated with primary antibody overnight at 4 °C and then with a secondary antibody for 1 h. Cell nucleus was subsequently stained with DAPI, and was imaged under a Laser scanning Confocal Microscopy (Leica Biosystems).

Lung tissues were washed in PBS and fixed in 10% PBS-buffered formalin for 24 h. Tissues were then embedded in optimal cutting temperature compound, and 5–6 μm sections were cut by Microtome Cryostat (Leica CM3050S, Leica Biosystems), and stained with antibody according to the protocol provided by the manufacturer. Cells, cultured on the microscope cover glass, were washed in PBS and fixed in 10% PBS-buffered formalin for 15 min, and then stained with antibodies according to the protocol provided by the manufacturer. The sections were examined using a laser scanning confocal microscopy (Leica Biosystems). Primary antibodies (1 : 200) were applied as follows: Rat anti-mouse C3 (Abcam ab11862), Rabbit anti-C3 (Abcam ab200999), Rat anti-mouse Nestin (Abcam ab81462), Rat anti-mouse Ly6G (Abcam ab25377), Rabbit anti-human C3 (Abcam ab97462), Mouse monoclonal anti-human Nestin (Abcam ab22035), Rabbit anti-Histone H3-cit (Abcam ab5103), and Rabbit anti-Myeloperoxidase (Abcam ab208670). The following secondary antibodies (1 : 1000) were used: Alexa Fluor 488 Goat anti-Rat IgG (Abcam ab150157), Alexa Fluor 488 Goat anti-Rabbit IgG (Abcam ab150077), Alexa Fluor 555 Donkey anti-Rabbit IgG (Beyotime A0453), Alexa Fluor 647 Goat anti-mouse IgG (Beyotime A0473), and Alexa Fluor 594 Goat anti-Rat IgG (Abcam ab150160).

Treated neutrophils were fixed in 4% PFA in PBS for 20 minutes at room temperature and washed with PBS. Nuclei were subsequently stained with Hoechst, and was imaged under a Laser scanning Confocal Microscopy (Leica Biosystems).