Kp 1 clone

2 μm-thick consecutive tissue sections were prepared from formalin-fixed and paraffin-embedded tissues, provided by the Pathology Department of the Humanitas Clinical and Research Center, and processed for immunohistochemistry. Briefly, after deparaffinization and rehydration, antigen retrieval was performed by heat treatment using EDTA buffer (0.25 mM, pH8, Dako) or citrate buffer (0.01 M, pH6, SIGMA-ALDRICH) in water bath at 98 °C for 20 min or pressure cooker. Endogenous peroxidases were blocked by incubation with 3% H₂O₂ for 15 min at room temperature, followed by incubation for 30 min with 2% BSA to block non-specific binding. The sections were then incubated with primary antibodies anti-human CD68 (Dako, KP-1 clone, diluted 1:1000), CD20 (Dako, L26 clone, diluted 1:200), CD8 (Dako, C8/144B clone, diluted 1:100), PD-1 (Abcam, NAT105 clone, diluted 1:50), CD45RO (Dako, UCHL1 clone, diluted 1:200), IL-17 (R&D, AF-317-NA, 1:500) for 1 h at room temperature, followed by incubation with the detection system MACH 1 (Biocare Medical) or Anti-Goat Polymer kit (Biocare Medical). Diaminobenzidine tetrahydrochloride (Biocare Medical) was used as chromogen. Nuclei were lightly counterstained with a freshly made hematoxylin solution (Dako). The sections were then washed in water, mounted and analysed under an optical microscopy.