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.