Anti inos

The sections were dewaxed, rehydrated, and then immersed into citrate buffer for antigen repair. After that, the sections were blocked with 2% bovine serum albumin and then incubated overnight at 4 °C with the following primary antibodies: anti-iNOS (1:200; Invitrogen, USA), anti-ionized calcium binding adaptor molecule-1 (Iba-1) (1:100, Abcam, USA), anti-Arg-1 (1:200, Santa Cruz Biotechnology, USA), followed by incubation with the secondary antibodies Alex Fluor® 488-labeled secondary antibody (green; 1:1000; Abcam), Alex Fluor® 647-labeled secondary antibody (red; 1:1000; Abcam) and Hoechst (blue; 1:1000) for 1 h at room temperature. Slides were observed by a fluorescent microscope (Olympus IX-71) equipped with a Canon EOS digital camera. The iNOS, Arg-1, and Iba-1 positive cells were separately counted using Image J software.

Fourteen-μm-thick aortic cross sections were obtained in a cryostat and were blocked with a 5% solution of bovine serum albumin (Sigma-Aldrich). Afterwards, sections were incubated with mouse monoclonal anti-eNOS (1:100; BD Biosciences, Franklin Lakes, NJ, USA, #610297), or a rabbit polyclonal anti-iNOS (1:50; ThermoFisher scientific, Waltham, MA, USA, #PA1-036) and anti-nNOS (1:100; ThermoFisher scientific, Waltham, MA, USA, #61-7000) antibodies at 37 °C, as described^{41 (link)}. After washing, slides were treated with an anti-mouse (#715-165-150) or anti-rabbit (#711-165-152) Cy3^™ secondary antibody (1:200; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA). Sections were mounted with fluorescence mounting medium (Code S3023; Dako, Agilent, Santa Clara, CA, USA). Images were captured with an Olympus FluoView 1000 confocal system (×20 objective; Olympus, Shinjuku, Tokio, Japan). Several fluorescent regions between elastin laminae of the smooth muscle layer, and within the endothelium were delineated and averaged using ImageJ 1.51j8 software. Then, the global average intensity in at least three sections per animal was obtained.

Skin samples were lysed with RIPA buffer (BOSTER) and PMSF (BOSTER) to extract protein. The concentration of protein was determined with a BCA protein assay kit (BOSTER). An equivalent amount of protein was loaded onto 10% SDS-PAGE gels (BOSTER), electrophoretically treated and then transferred to PVDF membranes (Millipore). The membranes were incubated with 5% nonfat milk in TBS-T for 2 h at room temperature, and then incubated with primary antibodies at 4 °C overnight. The membranes were incubated with secondary antibodies for 2 h at room temperature, and the protein bands were visualized using ECL reagents. Finally, the bands were photographed using gel image analysis system (Tanon Science & Technology Co., Shanghai, China) and the intensity of the bands was quantified in Gel-Pro analyzer software (Media Cybernetics). The details of the primary and secondary antibodies are as follows: anti-iNOS (1:1000, Thermo Fisher Scientific), anti-Arg-1 (1:500, BOSTER), and horseradish peroxidase-conjugated AffiniPure goat anti-rabbit IgG (1:2000, Jackson ImmunoResearch).

The tissue distribution of nuclear factor erythroid 2–related factor 2 (Nrf2), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) proteins were detected by IHC as described in a previous study [32 (link)]. After deparaffinization of tissue sections, anti-Nrf2 (Abcam, Cambridge, UK), anti-COX-2 (Cell Signaling Technology Inc., Danvers, MA, USA), anti-iNOS (Thermo Fisher Scientific Inc., Waltham, MA, USA) and goat alkaline phosphatase (AP) conjugated anti-rabbit IgG (1:200, Thermo Fisher Scientific Inc., Waltham, MA, USA), antibodies were sequentially treated onto these sections. Finally, the distribution of each protein in the retina was detected using stable diaminobenzidine (DAB) (Invitrogen Co., Waltham, MA, USA) and evaluated using the Leica Application Suite (Leica Microsystems).