Optimized Immunohistochemical Protocol

Immunohistochemical labeling was performed according to standard protocols on serial 2.5 μm thick sections from the original blocks or the TMA blocks. All cases were also stained for vimentin (clone V9, Dako, Glostrup, Denmark) and cytokeratin 8/18 (clone 5D3, Novocastra^TM, Leica Biosystems, Newcastle, U.K), which were used as control stains for tissue immunoreactivity and fixation, as well as identification of tumor cells. Tissue samples negative for the above antibodies (21 of 975, 2.2%) were excluded from the study. To assure optimal reactivity, immunostaining was applied 7 to 10 days after sectioning at the Laboratory of Molecular Oncology of the Hellenic Foundation for Cancer Research, Aristotle University of Thessaloniki School of Medicine. The staining procedures for vimentin, cytokeratin 8/18, HER2 (A0485 polyclonal antibody, Dako), estrogen receptor (ER, clone 6F11, Novocastra^TM, Leica Biosystems), progesterone receptor (PgR, clone 1A6, Novocastra^TM, Leica Biosystems) and Ki67 (clone MIB-1, Dako) were performed using a Bond Max^TM autostainer (Leica Microsystems, Wetzlar, Germany), as previously described in detail [33 (link)–35 (link)].