Anti her2 clone 4b5

For immunohistochemistry, 2 μm sections of formalin-fixed paraffin-embedded tumor specimens were mounted on poly-L-lysine-coated slides, and deparaffinized and rehydrated conventionally. Immunohistochemical stainings were performed on a Benchmark Ultra (Ventana, U.S.A.) automated stainer using the CC1-mild or CC1-st protocols for target retrieval and the anti-estrogen receptor α (clone SP1, Ventana, undiluted), anti-progesterone receptor α (clone 1E2, Ventana, undiluted), anti-HER2 (clone 4B5, Ventana, undiluted), anti-E-Cadherin (clone MSK 033, Zytomed, Berlin, Germany, 1:100) and anti-Ki-67 (clone SP6,Thermo Scientific, Germany, 1:100) antibodies. HER2 in situ hybridization was done as described. 20

We used 3-μm-thick tissue sections for the immunohistochemical (IHC) analysis of the estrogen receptor (ER), progesterone receptor, HER2, basal and myoepithelial markers (cytokeratin 5/6 and p63), and Ki-67. We used the following antibodies: anti-ER (clone 1D5, DAKO, Santa Clara, CA, USA; dilution 1: 50), antiprogesterone receptor (clone PgR636, DAKO; dilution 1: 50), anti-Ki-67 (clone MM1, Leica Biosystems Newcastle Ltd., Newcastle upon Tyne, UK; dilution 1: 100), anti-HER2 (clone 4B5, Ventana, Tucson, AZ, USA; prediluted), and anti-cytokeratin 5/6 and anti-p63 (based on a previous study) [21] . IHC analysis was optimized by evaluating serial sections with multiple antibody concentrations using a Discovery Automated Immunostainer (Ventana Medical Systems, Tucson, AZ, USA).
For HER2 testing, dual-color fluorescence in situ hybridization (FISH), using the PathVysion HER-2 DNA Probe Kit (PathVysion; Abbott Molecular, Des Plaines, IL, USA), and IHC assessment were used in accordance with the guidelines of the American Society of Clinical Oncology (ASCO) [22] . After counting the signals of HER2 and those of centromeric enumeration probe 17 (CEP17) under a fluorescence microscope, a HER2/CEP17 ratio ≥2.0 was defined as amplification.