EBER-in situ hybridization (ISH)

EBER (Epstein-Barr virus-encoded small RNAs) ISH was performed using a Ventana Benchmark ISH system (Ventana ISH iView kit, Ventana Corporation, Tucson, AZ, USA). Paraffin-embedded tissue sections were deparaffinized using EZ Prep buffer (Ventana Corporation), and then digested with Protease I for 4 min. Probes were added to the sample, and then denaturation was performed at 85°C (10 min), followed by hybridization at 37°C (1 hour). The probes labeled with fluorescein contained a cocktail of oligonucleotides dissolved in a formamide-based diluent. After hybridization, tissues were washed 3 times using 2x saline sodium citrate buffer at 57°C. Incubation with anti-fluorescein monoclonal antibody was performed for 20 min and then the alkaline blue detection kit (Ventana Corporation) was used according to the manufacturer's protocol. The slides were counterstained with nuclear fast red (Ventana Corporation) for 10 min.