Ariol sl 200

All of 28 FGFR2-positive tumors and 5 of FGFR2-negative tumors were examined FGFR2 gene amplification by FISH analysis. The area most strongly immunostained of tumor paraffin-embedded sections were subjected to FISH. Tumor sections were cut to 4 μm thickness, followed by deparaffinization with the pretreatment reagent (Abbott Molecular Inc., 2J06-30) at 80 ± 1 °C for 30 min. Protease digestion procedures were performed using the protease reagent (Abbott Molecular Inc., 2J08-32) at 37 ± 1 °C for 60 min. FGFR2/CEN10p Dual Color FISH Probe conjugated with Texas Red/FITC (GSPLab., Inc, GC018) were hybridized at 75 °C for 5 min, and 37 °C for 48 h (Supplement Fig. S2). After hybridization, the slides were washed in 2 × saline-sodium citrate/0.3% NP-40 at 72 °C for 5 min, and counterstained with PROLONG Gold antifade reagent with DAPI (Thermo Fischer Scientific, P36935). The specimens were examined with ARIOL SL-200 (Leica Biosystems). FGFR2/CEN10 signals of 40 tumor cell nuclei were counted and FGFR2/CEN10 ratio was calculated according to the evaluation method of The PATHVYSION HER-2 DNA Probe Kit (Abbott). An FGFR2/CEN10 ratio 2.0 or higher were defined as gene amplification positive.

FFPE tissue Sects. (5-µm thick) were deparaffinized and digested using standard processing methods for FISH. The PathVysion HER2 DNA Probe Kit (Abbott Molecular/Vysis) was used for the hybridization of tissue sections. Hybridization and counterstaining were performed according to the manufacturer’s instructions. Slides were imaged using an Applied Imaging system running Ariol SL200 (Leica Biosystems, Tokyo, Japan). At least 30 nuclei were evaluated, and the results were interpreted following the 2018 American Society of Clinical Oncology and the College of American Pathologists guidelines [15 (link)].