For immunohistochemistry, unstained 3 μm sections from FFPE tissue specimens were cut on the manual rotary microtome (AccuCut, Sakura, Torrance, USA). The immunohistochemical procedure was standardized using a series of positive and negative control reactions. Immunohistochemical stainings were performed according to well-known protocols [6 (link), 7 (link)]. We investigated expressions of proteins: CK7, cyclin D1, p16, survivin, CD138, Ki-67 and cleaved CASP3.
For primary antibodies such as rabbit monoclonal cytokeratin 7 (SP52), rabbit monoclonal cyclin D1 (SP4-R), mouse monoclonal p16 (E6H4) and mouse monoclonal Ki-67 (30-9) (Ventana Medical Systems) immunohistochemical staining was performed on the Benchmark GX Platform (Ventana Medical Systems, Tuscon, AZ, USA). We used visualization system UltraView DAB IHC Detection Kit (Ventana Medical Systems, Tuscon, AZ, USA) as recommended by the producer. Using the EnVision system detection (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA), immunohistochemical procedure was performed for monoclonal mouse Survivine (12C4), monoclonal mouse CD138 (MI15), (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) and CASP3 (Ab13847) (Abcam, Cambridge, UK).
For primary antibodies such as rabbit monoclonal cytokeratin 7 (SP52), rabbit monoclonal cyclin D1 (SP4-R), mouse monoclonal p16 (E6H4) and mouse monoclonal Ki-67 (30-9) (Ventana Medical Systems) immunohistochemical staining was performed on the Benchmark GX Platform (Ventana Medical Systems, Tuscon, AZ, USA). We used visualization system UltraView DAB IHC Detection Kit (Ventana Medical Systems, Tuscon, AZ, USA) as recommended by the producer. Using the EnVision system detection (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA), immunohistochemical procedure was performed for monoclonal mouse Survivine (12C4), monoclonal mouse CD138 (MI15), (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) and CASP3 (Ab13847) (Abcam, Cambridge, UK).
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