Quantitative Immunohistochemical Analysis

Expression levels of therapeutic targets such as HER2/neu, EGFR and MIB1 were determined using immunohistochemistry (IHC). The immunohistochemical stainings with anti-HER2/neu (A0785; 1:300, DAKO, Hamburg, Germany), anti-EGFR (pharmDx™-kit, DAKO) and anti-MIB1 (M7240; 1:100, DAKO) antibodies were performed on 3 μm consecutive sections using an automated slide processing system (Ventana DISCOVERY XT System, Ventana Medical Systems, Inc., Tucson, USA) in accordance with the manufacturer's instructions. IHC staining and analysis were performed as previously described [37 (link)]. MIB1 score was defined as Score 0 (0 - 20% of all nuclei were positive), Score 1 (20 – 50%), Score 2 (50 – 80 %) and Score 3 (80 - 100%).