Immunohistochemical staining for endothelial nitric oxide synthase and oxidative stress
Vascular expression of endothelial nitric oxide synthase was quantified using eNOS/NOS3 antibody (1:100 dilution, Thermo Fisher Scientific, USA), which is the key nitric oxide generating enzyme within the vascular endothelium. Vascular oxidative stress was measured using 3-Nitrotyrosine (3-NT) (1:100 dilution, Thermo Fisher Scientific, USA) as this is a specific marker for peroxynitrite production, the direct product of the reaction between superoxide to nitric oxide. Thoracic aorta was collected upon cull and fixed in 4% paraformaldehyde prior to sucrose saturation. The aortae were then paraffin embedded and 4 µM sections were cut. Aortic sections were subject to standard deparaffinisation and rehydration, followed by antigen retrieval (in 10mM citric acid, 0.05% Tween 20, pH 6.0) and blocking (blocking buffer; 10% horse serum, 10% FBS, 2% triton-X, 1xPBS to 50mL for 1hr). Sections were then incubated overnight with either eNOS of 3-NT primary antibodies at 4 ̵֯C. Excessive primary antibodies were removed by washing, the sections were then incubated at room temperature with the fluorescently labelled secondary antibody, Alexa 488 (1:200 dilution, Thermo Fisher Scientific, USA), then cover slipped using Fluoromount-GTM, with DAPI (Thermo Fisher Scientific, USA) prior to imaging on an Olympus slide scanner VS120-SS (Olympus, Japan). The expression of eNOS and 3-NT was quantified in the endothelial layer of the aortae using Olympus cellSens DimensionTM desktop software, calculating Object Area Fraction ROI (%) (version 1:18, Olympus Corporation). All analysis of immunofluorescence was completed in a blinded manner.