Epstein–Barr virus-encoded small RNA in-situ hybridization (EBER-ISH)

To confirm the location of EBV in tumor tissues, after real-time PCR was done, Epstein–Barr virus-encoded small RNA (EBER) was detected using in situ hybridization. FFPE tissue samples were cut into 3 μm sections and mounted on slides. A section of FFPE nasopharyngeal carcinoma tissue was used as the positive control. EBER PNA probe/FITC (DakoCytomation, Y5200, Glostrup, Denmark) and the Ab93705-mouse and rabbit specific HRP/AEC (ABC) Detection IHC Kit (Abcam, Boston, MA, USA) were used as a probe and detection system, respectively. After the tissue samples were deparaffinized, rehydrated, hydrogen peroxide block solution was applied. The EBER probe was applied to the tissue sections and incubated at 55 °C for 90 min followed by rabbit anti-FITC and biotinylated goat anti-polyvalent. Then streptavidin peroxidase was applied followed by AEC Single solution and counterstained with hematoxylin. A red color (positive) signal of EBV-infected cells could be observed under a light microscope and the EBV localization in the tumor tissue was confirmed by a pathologist.