Thermobrite

Slides were incubated for 10 min in HCl 0.1N/pepsin (0.5%) at 37 °C, and then washed in PBS. Slides were incubated in formaldehyde (1%) for 10 min, dehydrated in ethanol 70°, 90° and 100° (3 min each) and dried. Denaturation and hybridization were performed with a thermobrite (Leica Biosystems, Wetzlar, Germany). Denaturation was performed following manufacturer’s instructions: 10 min at 75 °C for ROS1 probe (zytolight SPEC ROS1 Dual-Color Break-Apart probe (PL101), zytovision, Bremerhaven, Germany), 4 min at 75 °C for ALK probe (Vysis LSI ALK Dual-Color Break-Appart, rearrangement probe, Abbott molecular, Des Plaines, IL, USA). Hybridization was performed overnight at 37 °C. Slides were washed in a buffered bath (saline–sodium citrate buffer 0.4Xat 72 °C for 2 min, then twice at room temperature for 30 s). Nuclei were stained with DAPI for 10 min at room temperature (DAPI II, Abbott, Chicago, IL, USA). Slides were mounted using FluorSave™ reagent (Merck Millipore, Burlington, USA) and observed using a fluorescence microscope (Leica, Wetzlar, Germany).

Tissue microarrays containing 295 DGC and 112 IGC samples were used for in situ miRNA hybridization. In situ hybridization was performed using a miRCURY LNA miRNA ISH kit (Qiagen) with miR-199a-5p (5′-ACAGGTAGTCTGAACACT-3′) and miR-199b-5p (5′-AACAGATAGTCTAAACACT-3′) probes. The U6 and scramble miRNA probes provided in the kit were used as positive and negative controls, respectively. All probes were labeled with double digoxigenin (DIG) and denatured at 90 °C for 4 min before use. After deparaffinization and hydration, the slides were incubated with proteinase K (20 μg/mL) at 37 °C for 5 min (miR-199a-5p) or 15 min (miR-199b-5p). Slides were hybridized with probes against miR-199a-5p (50 nM, at 50 °C) and miR-199b-5p (125 nM at 40 °C) overnight in a hybridizer (ThermoBrite, Leica Biosystems, Richmond, IL). For probe detection, slides were incubated with sheep anti-DIG-alkaline phosphatase antibody (1:100; Roche, Mannheim, Germany) at room temperature for 1 h. The remaining steps were performed in accordance with the manufacturer’s instructions. The intensity of miR-199a and miR-199b expression was graded as follows: 0, no staining; 1, mild tumor cell staining; 2, moderate tumor cell staining; and 3, strong tumor cell staining. miR-199a and miR-199b expression levels were classified as low (0 and 1) and high (2 and 3), respectively.