Fluorescence In Situ Hybridization Assessment of c-myc Gene Amplification in Breast Tumor Tissues
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This chapter details methods used for analysis of DNA copy-number changes in breast tumor tissues through the use of fluorescence in situ hybridization. The specific DNA probe described herein is the oncogene c-myc , although the tissue fluorescence in situ hybridization methodology presented is suitable for dual-color studies of most unique sequence and chromosome specific control probes.
The breast tumor tissue sections are first deparaffinized in a solvent and clearing agent, and then pretreated with a protease to allow the target DNA within the breast tissue cells to be uncovered. This allows the DNA to be available for hybridization with the labeled c-myc probe. The tissue sections are then analyzed to assure that appropriate digestion of cellular material has been attained. The tissues are then denatured. The c-myc probe and control probe for the centromere of chromosome 8 are commercially available as differentially labeled and are cohybridized to the tissue and sealed beneath a cover slip in a humid chamber. They are incubated for 12–16 h. The cover slip is then removed, and the section is postwashed in 2X saline sodium citrate at 72�C for 5 min and allowed to cool to room temperature in a detergent solution. The slides are then counterstained with 4′,6-diamidino-2-phenylindole, and a cover slip is applied. The slides are then viewed with fluorescence microscopy using filters that allow the c-myc and chromosome 8 signals to be visualized. If possible, 50 cells are counted, and the data are expressed as number of c-myc signals/number of chromosome 8 signals.
