Anti er clone sp1

For immunohistochemistry, 2-μm sections of formalin-fixed paraffin-embedded (FFPE) tumor specimens were mounted on poly-L-lysine-coated slides and deparaffinized and rehydrated conventionally. Immunohistochemical stainings were performed on a Benchmark Ultra (Ventana, USA) automated stainer using the CC1-mild or CC1-st protocols for target retrieval and the anti-CD10 (clone 56C6, Thermo Scientific, Germany, 1:10), anti-CD34 (clone QBEnd/10, Leica Biosystems, Germany, 1:50), anti CK5/14 (clone XM26/LL002, Zytomed Systems, Germany, 1:100), anti-CK8/18 (clone 5D3, Leica Biosystems, 1:100), anti-p63 (clone Y4A3, Zytomed Systems, 1:50), anti-p40 (rabbit polyclonal, PC373, Calbiochem, U.S.A., 1:1000), anti-smActin (clone 1A4, Dako, Denmark, 1:100), anti-EGFR (clone 2-1E1, Zytomed Systems, 1:200), anti-ER (clone SP1, Ventana, 1:1), anti-PR (clone 1E2, Ventana, 1:1), anti-HER2 (clone 4B5, Ventana), and anti-Ki-67 (clone SP6, Thermo Scientific, 1:100) antibodies. Besides characteristic histomorphology, at least two of the three myoepithelial markers (CK5/14, p63 or p40, CD10) being positive in more than 10 % of tumor cells were required for inclusion into the study.

The measurements of hormone receptors including ER and PR as well as HER2 and Ki67 were performed by the immunohistochemistry method according to the manufacturer’s indications. To define ER and PR status, immunostaining using anti-ER (clone SP1) and anti-PR (clone 1E2) pre-diluted monoclonal antibodies (Ventana Medical Systems, Tucson, AZ, USA) was applied. For the demonstration of the Ki67 antigen in the specimens, monoclonal mouse antibody (Autostainer Link 48, Agilent Technologies, Santa Clara, CA, USA) was used. For the semi-quantitative detection of HER2, the rabbit monoclonal primary antibody VENTANA anti-HER2/neu (clone 4B5) was used.
The immunostaining for ER, PR, and Ki67 was quantified and expressed as percentages of immunostained neoplastic cells. We used 20% as the threshold value to judge the level of Ki67 expression. For HER2, strong complete membrane staining in more than 10% of tumour cells was defined as positive expression (3+). Scores of 0 and 1 were established as negative expression, and all tumour cells with a 2+ score were further tested by fluorescence in situ hybridisation [19 ]. Based on the expression of molecular determinants, all subjects were classified according to the St. Gallen 2013-15 recommendations.