Immunofluorescence
Automated double immunofluorescence was conducted using the Leica Bond BX staining system. Paraffin-embedded tissues were sectioned at 5 μm and baked at 58 °C for 1 h. Slides were loaded in Leica Bond and staining was performed as follows. Samples were pretreated with EDTA-based epitope retrieval ER2 solution (Leica, AR9640) for 20 min at 95 °C. The double antibody staining and detection were conducted sequentially. Primary antibodies against CD3 (0.6 µg ml