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Immunofluorescence Microscopy to Assess Enzymes Controlling Nitric Oxide Availability and Microvascular Blood Flow in Muscle


Anton Wagenmakers, Ph.D., School of Sport and Exercise Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.


Please cite this paper as: Cocks M, Shepherd SO, Shaw CS, Achten J, Costa ML, Wagenmakers AJM. Immunofluorescence microscopy to assess enzymes controlling nitric oxide availability and microvascular blood flow in muscle. Microcirculation 19: 642–651, 2012.


Objective:  The net production of NO by the muscle microvascular endothelium is a key regulator of muscle microvascular blood flow. Here, we describe the development of a method to quantify the protein content and phosphorylation of endothelial NO synthase (eNOS content and eNOS ser1177 phosphorylation) and NAD(P)H oxidase expression.

Methods:  Human muscle cryosections were stained using antibodies targeting eNOS, p-eNOS ser1177 and NOX2 in combination with markers of the endothelium and the sarcolemma. Quantitation was achieved by analyzing fluorescence intensity within the area stained positive for the microvascular endothelium. Analysis was performed in duplicate and repeated five times to investigate CV. In addition, eight healthy males (age 21 ± 1 year, BMI 24.4 ± 1.0 kg/m2) completed one hour of cycling exercise at ∼65%VO2max. Muscle biopsies were taken from the m. vastus lateralis before and immediately after exercise and analyzed using the new methods.

Results:  The CV of all methods was between 6.5 and 9.5%. Acute exercise increased eNOS serine1177 phosphorylation (fold change 1.29 ± 0.05, < 0.05).

Conclusions:  These novel methodologies will allow direct investigations of the molecular mechanisms underpinning the microvascular responses to insulin and exercise, the impairments that occur in sedentary, obese and elderly individuals and the effect of lifestyle interventions.