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Journal of Histochemistry and Cytochemistry, Vol. 47, 1395-1404, November 1999, Copyright © 1999, The Histochemical Society, Inc.

ARTICLE

Immunocytochemical Detection of Ornithine Decarboxylase

Raymond G. Schippera, Nadine Romainb, Adrianus A. Ottena, Jing Tanc, Will P. Langea, and Albert A. J. Verhofstada
a Department of Pathology, University Hospital Nijmegen, Nijmegen, The Netherlands
b IEEM-NMC Biochemistry Lab, Presbyterian Hospital of Dallas, Dallas, Texas
c Biology Convention Center, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas

Correspondence to: Albert A. J. Verhofstad, Dept. of Pathology, University Hospital Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands.

Ornithine decarboxylase (ODC), a regulatory enzyme of polyamine biosynthesis, is involved in cell growth and differentiation. Lack of information about the exact cellular and subcellular localization of ODC is one of the main obstacles to precise interpretation of the biological roles of the ODC/polyamine system. Here we describe the development and optimization of an immunocytochemical method to detect ODC in cells and tissues. For this purpose a monoclonal antibody (MP16-2) against a defined epitope of ODC protein was developed. Specificity of the antibody for ODC was substantiated by Western blotting and ELISA analysis using cell and tissue homogenates. In cultured cells, optimal staining results were obtained after fixation with crosslinking fixatives followed by permeabilization with methanol. In rat tissues, ODC immunoreactivity was best preserved in paraffin sections fixed with Bouin's fixative. Antigen retrieval using SDS and citrate buffer substantially increased ODC immunostaining and decreased background staining. Localization studies of ODC in different cell lines showed that strongest staining for ODC was found in the nucleoplasm of mitotic cells, whereas confluent cells showed moderate perinuclear staining. Immunocytochemical studies of various rat tissues showed high cytoplasmic immunostaining of ODC in epithelial cells of kidney, prostate, and adrenal medulla of testosterone-treated rats, in glandular epithelium of small intestine, and in pancreas of neonatal and adult rats. (J Histochem Cytochem 47:1395–1404, 1999)

Key Words: ornithine decarboxylase, monoclonal antibody, immunocytochemistry, tissue fixation, cell lines, rat


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