Ir702

Slides were deparaffinised, rehydrated and immersed in phosphate buffered saline solution (10 mM, pH 7.2). Tissue epitopes were demasked through revitalisation in TRIS-EDTA retrieval solution (10 mM TRIS, 1 mM EDTA, pH 9,0) at 98 °C for 20 min in Dako PT Link (Dako, Glostrup, Denmark). The slides were subsequently incubated for 1 h at room temperature with primary mouse monoclonal antibody against β-catenin (IR702, Ready-to-Use, Dako) and immunostained using anti-mouse/anti-rabbit secondary antibody (EnVision FLEX/HRP, Dako) for 30 min at room temperature. The reaction was visualised by diaminobenzidine substrate-chromogen solution (DAB, Dako) which was applied for 5 min. Ultimately, the slides were counterstained with hematoxylin. Non-neoplastic testicular tissue was used as a positive control and the same tissue without incubation in primary antibody represented the negative control. Representative images were captured with Olympus BX40 microscope (Olympus Corporation, Tokyo, Japan) and Canon EOS 1000D (Canon Inc., Tokyo, Japan).

The 4-μm-thick sections were subjected to immunostaining with a TFE3 antibody (ZA-0657; ZSGB-BI) and a β-catenin antibody (IR702; Dako) using the Dako Omnis automated staining platform. TFE3, a monoclonal antibody was diluted to a ratio of 1:100. Animal serum and buffer were used as a negative control instead of primary antibody. Immunoreaction of TFE3 was performed on the DAKO automated immunostainer. The antibody was optimized using Ventana DAB detection kit (Ventana Medical Systems) and standard quality control procedures were carried out. The detection of TFE3 was performed following heat-induced antigen retrieval using ER2 antigen retrieval buffer. Xp11.2 renal cell carcinomas were used as positive control, while negative cell controls were also used.