Novocastra

Immunohistochemical labeling was performed according to standard protocols on serial 2.5 μm thick sections from the original blocks or the TMA blocks. To assure optimal reactivity, immunostaining was applied 7–10 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 HER2 (A0485 polyclonal antibody, Dako, Glostrup, Denmark), estrogen receptor (ER, clone 6F11, Novocastra™, Leica Biosystems, Newcastle, U.K), progesterone receptor (PgR, clone 1A6, Novocastra™, Leica Biosystems) and Ki67 (clone MIB-1, Dako) were performed using a Bond Max™ autostainer (Leica Microsystems, Wetzlar, Germany), as previously described in detail [25 (link)–28 (link)]. The staining procedures for OPN (clone OP3 N, code NCL-O-PONTIN, Novocastra™, Leica Biosystems) were also performed using a Bond Max™ autostainer, as previously described [29 (link)].

Tissue microarrays (TMAs) were constructed from FFPE curettage and hysterectomy biopsies as previously described [23 (link), 24 (link)]. The TMA slides (5 µm) were dewaxed in xylene and rehydrated in ethanol before antigen retrieval in target retrieval solution at pH 9 or pH 6 for 15 min in microwave followed by peroxidase block (S2023, Dako, Glostrup, Denmark) for 8 min at room temperature. Sections were incubated for 60 min at room temperature with anti-MSH6 monoclonal mouse antibody (1:25, NCL-L-MSH6, NovocastraTM, Leica Biosystems Newcastle, United Kingdom) or anti-PMS2 monoclonal mouse antibody (1:25, PMS2-L-CE; NovocastraTM, Leica Biosystems Wetzlar, Germany) or incubated for 30 min at room temperature with anti-MLH1 monoclonal mouse antibody (1:100; NCL-L-MLH1; NovocastraTM, Leica Biosystems Newcastle, UK) or anti-MSH2 monoclonal mouse antibody (1:50; NCL-l-MSH2–612; NovocastraTM, Leica Biosystems Newcastle, UK). All sections were incubated for 30 min with a secondary anti-mouse antibody (Agilent Technologies, Santa Clara, USA). Staining was visualised with diaminobenzidine peroxidase (DAB) (EnVision detection system, K3468, Dako, Glostrup, Denmark). Sections were counterstained with hematoxylin (S3301, Dako, Glostrup, Denmark) before dehydration and mounting.

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)].