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JMD 2000, Vol. 2, No. 2
Copyright © 2000 American Society for Investigative Pathology & Association for Molecular Pathology

Concomitant Oncoprotein Detection with Fluorescence in Situ Hybridization (CODFISH)

A Fluorescence-Based Assay Enabling Simultaneous Visualization of Gene Amplification and Encoded Protein Expression

Raymond R. Tubbs^*, James Pettay^*, Pat Roche, Mark H. Stoler, Robert Jenkins, Jon Myles^* and Thomas Grogan

From the Departments of Clinical and Anatomic Pathology, * the Cleveland Clinic Foundation, Cleveland, Ohio; the Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota; the Department of Pathology, University of Arizona School of Medicine, Tucson, Arizona; and the Robert E. Fechner Laboratory of Surgical Pathology, University of Virginia School of Medicine, Charlottesville, Virginia

We sought the validation of a three-color fluorescence-based system that simultaneously profiles Her-2/neu oncogene copy by fluorescence in situ hybridization (FISH) and Her-2/neu encoded protein by the use of a versatile alkaline phosphatase chromogen fast red K in either fluorescence or bright-field mode. Nuclei were counterstained with DAPI. Nineteen infiltrating ductal carcinomas of breast were comprehensively evaluated for Her-2/neu amplification/overexpression by direct and indirect FISH using digoxigenin (DigFISH) and direct fluorescently labeled probes, autoradiographic RNA:RNA in situ hybridization, and immunohistochemistry using monoclonal antibody CB11. CODFISH results correlated well with DigFISH, direct-label FISH, mRNA expression, and oncoprotein expression as assessed with CB11, and enabled simultaneous visualization of gene copy and protein. In addition, qualitative immunohistochemistry may be followed by CODFISH gene copy enumeration to clarify ambiguous cases.

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