A1 rs inverted laser scanning confocal microscope

Frozen sections of the aortas (7 μm thick) were fixed with cold 100% methanol and incubated for 2 h at room temperature in blocking buffer (5% of albumin in PBS). Tissue sections were then incubated overnight (4°C) with a mouse monoclonal antibody against the phosphorylated-(Ser 1179)-eNOS (1 : 50, Santa Cruz Biotechnology) or a rabbit polyclonal antibody against phosphorylated-(Ser473)-Akt (p-Akt) protein (1/500, Cell Signaling). Three washes were followed by incubation (1 h, at room temperature, in the dark) with the secondary mouse or rabbit fluorescent Alexa fluor-647-conjugated antibody (1 : 500, Molecular Probes). A Nikon A1-RS inverted laser scanning confocal microscope was used for the optical sectioning of the tissue. Digital image recording was performed using the NIS element software. Images were analysed and processed by Fiji software.

Frozen sections of aorta (7 μm thick) on glass slides were used to the in situ detection of superoxide anion (O₂⁻) with the oxidative fluorescent dye dihydroethidine (DHE, Sigma-Aldrich) as previously described by Miller et al. [13 (link)]. DHE oxidizes to EtBr in the presence of O₂⁻ and shows a red fluorescence.
In another set of experiments, the vessel frozen sections were fixed with cold 100% methanol and incubated (2 h at room temperature) in blocking buffer (5% nonfat dry milk in PBS). Tissue sections were then incubated overnight (4°C) with either a mouse monoclonal antibody antinitrotyrosine (1 : 100, Upstate Biothechnology), a monoclonal murine anti-iNOS (1 : 50, BD Transduction Laboratories), and a polyclonal NF-κB p65 antibody (1 : 100, Cell Signaling), for protein nitrotyrosine, iNOS, or NF-κB p65 immunostaining, respectively. Three washes were followed by incubation (1 h, at room temperature, in the dark) with Alexa fluor-647 anti-mouse or anti-rabbit secondary antibody (1 : 500, Molecular Probes). A Nikon A1-RS inverted laser scanning confocal microscope was used for the optical sectioning of the tissue. Digital image recording was performed using the NIS element software. Images were analyzed and processed by Fiji software.