Immunofluorescence microscopy to assess enzymes controlling nitric oxide availability and microvascular blood flow in muscle

Cocks, Matthew, Shepherd, Sam O., Shaw, Christopher S., Achten, Juul, Costa, Matthew L. and Wagenmakers, Anton J.M. 2012, Immunofluorescence microscopy to assess enzymes controlling nitric oxide availability and microvascular blood flow in muscle, Microcirculation, vol. 19, no. 7, pp. 642-651, doi: 10.1111/j.1549-8719.2012.00199.x.

Attached Files
Name Description MIMEType Size Downloads

Title Immunofluorescence microscopy to assess enzymes controlling nitric oxide availability and microvascular blood flow in muscle
Author(s) Cocks, Matthew
Shepherd, Sam O.
Shaw, Christopher S.ORCID iD for Shaw, Christopher S.
Achten, Juul
Costa, Matthew L.
Wagenmakers, Anton J.M.
Journal name Microcirculation
Volume number 19
Issue number 7
Start page 642
End page 651
Total pages 10
Publisher Wiley
Place of publication Chichester, Eng.
Publication date 2012-10
ISSN 1073-9688
Keyword(s) eNOS
NAD(P)H oxidase
muscle microvascular endothelial function
immunofluorescence microscopy
Blood Flow Velocity
Endothelium, Vascular
Gene Expression Regulation, Enzymologic
Microscopy, Fluorescence
Middle Aged
Nitric Oxide
Nitric Oxide Synthase Type III
Rats, Wistar
Summary 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, p < 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.
Language eng
DOI 10.1111/j.1549-8719.2012.00199.x
Field of Research 111699 Medical Physiology not elsewhere classified
Socio Economic Objective 970111 Expanding Knowledge in the Medical and Health Sciences
HERDC Research category C1.1 Refereed article in a scholarly journal
ERA Research output type C Journal article
Copyright notice ©2012, Wiley
Persistent URL

Document type: Journal Article
Collections: Faculty of Health
School of Exercise and Nutrition Sciences
Connect to link resolver
Unless expressly stated otherwise, the copyright for items in DRO is owned by the author, with all rights reserved.

Version Filter Type
Citation counts: TR Web of Science Citation Count  Cited 12 times in TR Web of Science
Scopus Citation Count Cited 11 times in Scopus
Google Scholar Search Google Scholar
Access Statistics: 180 Abstract Views, 1 File Downloads  -  Detailed Statistics
Created: Mon, 28 Nov 2016, 15:04:39 EST

Every reasonable effort has been made to ensure that permission has been obtained for items included in DRO. If you believe that your rights have been infringed by this repository, please contact