Concomitant oncoprotein detection with fluorescence in situ hybridization (CODFISH) - A fluorescence-based assay enabling simultaneous visualization of gene amplification and encoded protein expression

We sought the validation of a three-color fluorescence-based system that si multaneously profiles Her2/neu oncogene copy by fluorescence in situ hybrid ization (FISH) and Her-2/neu encoded protein by the use of a versatile alka line phosphatase chromogen fast red K in either fluorescence or bright-fiel d mode. Nuclei were counterstained with DAPI, Nineteen infiltrating ductal carcinomas of breast were comprehensively evaluated for Her-2/neu amplifica tion/over-expression 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. CO DFISH results correlated well with DigFISH, direct-label FISH, mRNA express ion, and oncoprotein expression as assessed with CB11, and enabled simultan eous visualization of gene copy and protein. In addition, qualitative immun ohistochemistry may be followed by CODFISH gene copy enumeration to clarify ambiguous cases.