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Improved Immunohistochemical Method for Detecting Hypoxia Gradients in Mouse Tissues and Tumors

Michael K. Samoszuk, James Walter and Eugene Mechetner

Pathology Department, University of California, Irvine, California (MKS), and Chemicon International, Inc., Temecula, California (JW,EM)

Correspondence to: Michael Samoszuk, Pathology Department, University of California, Irvine Medical Center, Building 10, Route 40, 101 The City Drive, Orange, CA 92868. E-mail msamoszu{at}uci.edu

We describe an improved immunohistochemical procedure for detecting regions of hypoxia in normal organs and tumors in mice. The method employs a primary fluorescein-conjugated mouse monoclonal antibody directed against pimonidazole protein adducts that are created in hypoxic tissues and a secondary mouse anti-fluorescein antibody that is conjugated to horseradish peroxidase. Using these reagents, we clearly visualized the regions of relative hypoxia in implanted tumors in mice as well as in normal organs such as liver and kidney. Significantly, the resulting tissue sections were remarkably free of the background staining that is characteristically observed when rodent antibodies are used to detect antigens in rodent tissues. (J Histochem Cytochem 52:837–839, 2004)

Key Words: immunohistochemistry • hypoxia • monoclonal antibodies

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