title: A novel immunohistochemical semiquantitative technique for endothelial constitutive nitric oxide synthase immunoreactivity in rat coronary artery.
abstract: It has been difficult to quantify protein production in small pathological specimens by conventional techniques. We describe a new method for semiquantification of immunohistochemical staining, which involves application of the enzyme-labeled avidin (LAB) technique, coupled with an ultra-sensitive and fast chemiluminescent substrate for alkaline phosphatase. The entire procedure can be completed in less than 3 hr. The final step involves X-ray film exposure for 30 min, and the optical density of the subsequent images is examined with a microcomputer imaging device. The optical densities are translated into relative protein concentrations by a reference standard curve, obtained via an immunoblot. To establish a model for semiquantification of endothelial constitutive nitric oxide synthase (eNOS) protein, we compared the coronary arteries of WKY rats fed a normal chow diet to the coronary arteries of WKY rats fed a cholesterol diet. Using this technique, we have found a relative 130-fold decrease in eNOS in the cholesterol-fed group compared to the normal chow-fed group.
authors.affiliation: Vascular Biology Unit, Department of Cardiac Surgery, University of Melbourne Austin Hospital, Heidelberg, Australia.
authors.forename: A
authors.initials: A
authors.lastname: Zulli
authors.forename: J J
authors.initials: JJ
authors.lastname: Liu
mesh_terms: D000818:Animals; D001158:Arteries / Q000201:enzymology; D002791:Cholesterol, Dietary / Q000008:administration & dosage*; D003331:Coronary Vessels / Q000201:enzymology*; D004730:Endothelium, Vascular / Q000201:enzymology*; D007091:Image Processing, Computer-Assisted; D015151:Immunoblotting; D007150:Immunohistochemistry / Q000379:methods*; D008163:Luminescent Measurements; D019001:Nitric Oxide Synthase / Q000032:analysis*; D051381:Rats; D011921:Rats, Inbred WKY; D012015:Reference Standards
chemical_list: D002791:Cholesterol, Dietary; D019001:Nitric Oxide Synthase