In situ analysis of lung metastasis

In situ FISH analysis was performed by the University of Colorado Denver Cytogenetics Core. Briefly, unstained slides with formalin-fixed, paraffin-embedded tissue sections were subjected to a dual-color FISH assay using the Human/Mouse probe set. This probe set was prepared by labeling 1 µg of Human Cot-1 DNA and Mouse Cot-1 DNA (Invitrogen) respectively with SpectrumRed and SpectrumGreen conjugated dUTPs (Abbott Molecular) using the Vysis Nick translation kit (Abbott Molecular), according to manufacturer’s instructions. The labeled DNAs were ethanol precipitated with herring sperm DNA as carrier (1:50) and each pellet was diluted to a final concentration of 50 ng/µl with tDenHybTM-2 hybridization buffer (Insitus Biotechnologies cat. no D102). The probe set was prepared such that it contained 100ng of each DNA per 4.5 µl and this volume was used on every 113mm2 hybridization area. The FFPE specimens were analyzed in an epifluorescence microscope using single interference filters sets for green (FITC), red (Texas red), blue (DAPI), and dual (red/green) and triple (blue, red, green) band pass. Each section was entirely examined under low magnification and selected areas were investigated under high magnification objectives.