4 6 diamidino 2 phenylindole staining

Human pulmonary microvascular endothelial cells (hPMVEC; PromoCell, Heidelberg, Germany), used at passages 4–6, were grown to confluence on glass slides, incubated with VT (300 ng/ml) or sterile PBS as a control for 90 minutes and then stimulated with LPS (0.1 μg/ml) from Escherichia coli (Sigma-Aldrich, Steinheim, Germany), PLY (0.25, 0.5, 0.75 μg/ml) [34 (link)], or PBS. After 30 minutes, cells were fixed in 3% formalin, and the tight junction protein vascular endothelial (VE)-cadherin (1:400, goat antihuman VE-cadherin; Santa Cruz Biotechnology, Santa Cruz, CA, USA; secondary antibody 1:8000, Alexa Fluor 488 F(ab′)₂ rabbit antigoat IgG (H + L), Invitrogen, Darmstadt, Germany) and actin fibers (1:2000; phalloidin Alexa 546-labeled; Invitrogen) were stained to evaluate cell morphology [35 (link)]. 4′,6-diamidino-2-phenylindole staining (Sigma-Aldrich) was used to visualize cell nuclei. The immunofluorescence of the cells was analyzed by confocal microscopy using an LSM 780 microscope with ZEN 2011 software (objective: Plan Apochromat × 63/1.40 oil; Carl Zeiss Microscopy, Jena, Germany).

Eyes (n=6) in optimal cutting temperature compound were cut into 6 µm sections (RM2165; Leica, Wetzlar, Germany) following being flash frozen in liquid nitrogen. The cryosections were fixed in ice-cold acetone for 10 min, washed in PBS three times and blocked with 1% bovine serum albumin (BSA) in PBST for 30 min at room temperature. Subsequently, primary goat anti-8-hydroxyguanine (8-OHdG, a classical DNA oxidative product) polyclonal antibody (1:100; cat. no. ab10802; Abcam, San Francisco, CA, USA) in 1% BSA in PBST were added to the samples and incubated in a humidified chamber overnight at 4°C. Following three washes in PBS, the sections were incubated with tetramethyl rhodamine isothiocyanate-conjugated rabbit anti-goat IgG secondary antibody (1:200; cat. no. BA1091; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) for 1 h in the dark at 37°C, followed by 4′,6-diamidino-2-phenylindole staining (0.1 µg/ml; Sigma-Aldrich, St. Louis, MO, USA) and another three washes in PBS. Coverslides were mounted and the immunoreactivity of 8-OHdG was detected under a fluorescence microscope (DFC500; Leica Microsystems, Renens, Switzerland).

The cell cycle was analyzed using flow cytometry (Cell Lab Quanta SC; Beckman Coulter, Miami, FL, USA) with 4′,6-diamidino-2-phenylindole staining (Sigma-Aldrich, St. Louis, MO, USA), as described in a previous study (12 (link)). Measurements were repeated independently three times.