Anti enos sc 654

RPE layers were prepared form 4‐week‐old wild‐type, PEDF−/−, and TSP1 −/− mice. Eyes were fixed in 4% PFA in PBS for 2 h. Following fixation, RPE, choroid, and sclera complex was prepared by removing the retina and cutting the optic nerve from the posterior portion of the eyes. The remaining cup was washed with PBS three times for 10 min, and blocked in blocking solution (50% FCS, 20% normal goat serum; NGS, in PBS containing 0.01% Triton 100X) at RT for 1 h. After blocking samples were washed with PBS three times for 10 min and incubated with primary antibodies including anti‐iNOS, anti‐HNE (Cell Signaling), anti‐eNOS (SC‐654) (Santa Cruz), and anti‐ZO‐1 (Life Technology) in PBS containing 20% FCS, 20% NGS, and 0.01% TX‐100 overnight at 4°C. Samples were then washed with PBS and incubated with specific Cy3‐conjugated secondary antibody (1:800; Jackson ImmunoResearch) in PBS containing 20% FCS, 20% NGS, and 0.01% TX‐100 at room temperature for 2–4 h. Following incubation, samples were washed five times with PBS, flattened by four radial cuts, mounted on the slide, and examined using a fluorescence microscope (Carl Zeiss Optical) and the images were taken in digital format.

Western blotting analysis was performed from the homogenized frozen biopsy according to Di Filippo et al. [19 (link)]. We used (i) primary anti-SIRT1 antibody (1 : 1000, Abcam, Cambridge, UK), (ii) specific monoclonal antibody directed against FOXO-1 (1 : 500, Millipore, California, USA), (iii) anti-MnSOD primary antibody (1 : 800, Millipore, California, USA), (iv) anti-β-actin monoclonal antibody (1 : 1000, Sigma-Aldrich), and (v) anti-eNOS sc-654 (1 : 500, Santa Cruz Biotech, USA) with an enhanced chemiluminescence detection reagent (ECL), quantified by densitometry using a BioRad ChemiDoc MP Imaging system. The following secondary antibodies were used: goat anti-mouse (1 : 1000, Santa Cruz Biotech, USA) and goat anti-rabbit (1 : 1000, Santa Cruz Biotech, USA). The deacetylase activity of SIRT1 was measured by means of a commercial fluorometric kit (Abcam, Cambridge, UK) and normalized by protein content.