Pepsin solution

Fluorescence in situ hybridization (FISH) of miR-214 was performed on 5 µm tissue sections of NPC following the manufacturer's instructions. Briefly, sections were at 59°C 2 h to attach cores to the silane-coated slide. Then, sections were de-paraffinised with xylene two times for 5 minutes each, rehydrated with ethanol (100 - 50 - 25% for 5 min each), and treated with DEPC water for 1 min. Subsequently, sections were treated with pepsin solution (1.3 mg/ml) (Dako, Glostrup, Denmark) at 37°C for 30 min. Following a post-fixation step in 4% paraformaldehyde (PFA), LNA-miR-214 detection probe (Exiqon, Vedbek, Denmark) or LNA-control (Exiqon, Vedbek, Denmark) was hybridized to the sections at 58°C for 4 hours carried out in a Hybrite (Abbott Laboratories, Shanghai, China). After post-hybridization wash, sections were incubated with anti-DIG-HRP (Roche, Shanghai, China) in a heater at 37°C for 30 min. Slides were washed by TNT buffer for 15 min at room temperature (RT) and incubated with Fluorophore TSA Reagent working solution for 10 min at RT. The slides were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) (Roche Diagnostics, Shanghai, China) and visualized the nuclei and glass mounting by fluorescence microscopy with a Jenoptik camera and VideoTesT-FISH 2.0 software (GP Medical Technologies Ltd, Beijing, China).