Lsi fgfr2 spectrum orange probe

Tumor sections were cut to 1-μm thickness and deparaffinized by incubation for 30 minutes with the pretreatment reagent (Abbott, 30-801250) at 80°C. Protease digestion was achieved by treatment with the protease reagent (Abbott, 30-801255) for 20 minutes at 37°C. FGFR2 probes (LSI FGFR2 Spectrum Orange Probe, 08N42-020) and CEP 10 (Spectrum Green Probe, 06J37-020) from Vysis (Abbott Molecular, IL) were hybridized at 73°C for 5 minutes and then at 37°C for 20 hours. After hybridization, slides were washed in 2× saline-sodium citrate/0.3% NP-40 at 72°C for 5 minutes, air dried, and counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) I and DAPI II (Abbott Molecular). Slides were then examined under a fluorescence microscope equipped with Spectrum Texas Red with isothiocyanate and DAPI filters. The FGFR2/CEP 10 ratio was estimated after counting at least 50 tumor cell nuclei. For all samples evaluated with FISH, IHC-negative stained areas in the tumors were also evaluated to determine the specificity of the IHC test. An FGFR2/CEP 10 ratio higher than 2.0 was interpreted as gene amplification positive. If the FGFR2 copy number was greater than 4 in the absence of gene amplification, FGFR2 polysomy was assumed.