Fluorescence in situ hybridization

The FISH analysis was performed on formalin-fixed, paraffin-embedded gastric cancer tissues and matched non-cancerous tissues using the EHF DNA probe/CEN11 probe mixture (Exon Biotechnology Inc, Guangzhou, P.R. China). Briefly, the paraffin-embedded tissue slides were deparaffinized through xylene, and rehydrated in an ethanol series (100, 85 and 70%), and treated with protenase K solution (200 μg/ml) and pepsin (0.005% in 0.01 M HCl solution) at 37 °C, respectively. The slides were then dehydrated in an ethanol series (70, 85 and 100%), and the probe mixture was added to the slides and immediately covered by coverslips and sealed the edges with rubber cement. The slides were subsequently denatured at 85 °C for 5 minutes and incubated at 37 °C overnight. After hybridization, the slides were washed in 2 × SSC, 2 × SSC/0.1% NP-40 buffer at 37 °C for 5 min each, and were counterstained with DAPI antifade solution. FISH signals in 20–30 cells for each specimen were counted, and the criteria for gene amplification were defined when FISH signals were detected by tested probes compared with control probes ≥1.5. Fluorescence images were captured with Olympus IX71 microscope (Olympus, Tokyo, Japan), which enables simultaneous detection of both FITC and Texas Red fluorescence. The color mergence was performed using ImageJ image software (ImageJ version 1.44p, NIH, MD).