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Journal of Histochemistry and Cytochemistry, Vol. 46, 257-262, Copyright © 1998 by The Histochemical Society, Inc.

ARTICLE

A Novel Immunohistochemical Semiquantitative Technique for Endothelial Constitutive Nitric Oxide Synthase Immunoreactivity in Rat Coronary Artery

Anthony Zullia and James J. Liua
a Vascular Biology Unit, Departments of Cardiac Surgery and Medicine, University of Melbourne Austin Hospital, Heidelberg, Australia

Correspondence to: James J. Liu, Vascular Biology Unit, Depts. of Cardiac Surgery and Medicine, U. of Melbourne Austin Hospital, Heidelberg VIC 3084, Australia.

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. (J Histochem Cytochem 46:257—262, 1998)

Key Words: Nonradioactive, protein semiquantification, eNOS, immunohistochemistry, coronary artery, cholesterol diet, chemiluminescence


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