Fluoview fv1000 laser scanning confocal system

The translocation of the NF-κB p65 subunit was activated by treatment with 1 μg/ml of LPS for 24 h and then presented with ZGC for another 48 h. Then, cells were fixed for 10 min in 4% methanol-free paraformaldehyde. Antibodies were diluted in PBS containing 2.5% fetal bovine serum and 0.1% Triton X-100 (Sigma). The NF-κB p65 subunit antibody was diluted at 1:500 in PBS. The samples were incubated overnight in a cold room. Then, 1: 1,000 dilution of the secondary FITC-conjugated F(ab’)2 fragment donkey anti-rabbit IgG antibody (Jackson Laboratories, West Grove, PA) was added and incubated at room temperature for 3 h in the dark. After incubation, the cells were washed three times with PBS. Before performing the assay, a 1:5,000 dilution of DAPI was added to the nucleus. Fluorimetric measurements were performed using an Olympus FluoView FV1000 laser scanning confocal system (Olympus America, Melville, NY) mounted on an inverted Olympus microscope equipped with a 63× objective.

After organ culture, some aortic rings were frozen in optimal cutting temperature compound (OCT; Sakura Finetek, Torrance, CA, USA) and later sliced to 10 μm sections by the CryoStar™ NX70 Cryostat (Thermo Fisher Scientific, Billings, MT, USA). ROS generation in the cross sections of mouse aortas were measured by DHE fluorescence staining, as previously described [31 (link)]. In brief, aortic cross sections were incubated with DHE (5 μmol L⁻¹; Invitrogen) at 37 °C for 15 min in normal physiological saline solution (NPSS) containing (in mmol L⁻¹): 140 NaCl, 5 KCl, 5 HEPES, 1 MgCl₂, 1 CaCl₂, and 10 glucose. Subsequently, the cross sections were rinsed thrice in NPSS. Fluorescent signals (DHE: excitation: 515 nm, emission: 585 nm; elastin autofluorescence: excitation: 488 nm, emission: 520–535 nm) were detected by the Olympus Fluoview FV1000 laser scanning confocal system (Olympus, Shinjuku City, Tokyo, Japan). DHE fluorescence signal was presented in real numbers.

ROS measurement in en face endothelium and cross-sectional area of rat aortae was performed as described [16 (link), 17 (link)]. Some aortae were freshly prepared for en face staining of ROS. Some aortae were embedded in OCT compound (Tissue-Tek), frozen in liquid nitrogen, and then cut into sections of 10-µm thickness on cryostat (Shandon). Fresh aortae or frozen sections of aortae were incubated for 20 min in 5 µM dihydroethidium (DHE; Molecular Probes)-containing PBS at 37 °C. Fluorescence was observed by Fluoview FV1000 laser scanning confocal system (Olympus, Tokyo, Japan; 515-nm excitation; 585-nm long-pass filter). DHE fluorescence intensity was analyzed by Fluoview FV10-ASW1.5 software. For each section, a square region with an area of 80 μm × 80 μm was selected for analysis. The summarized data represent the fold change in fluorescence intensity relative to that in control rat aortae [14 (link)].