Ki 67 30 9

After steam-heat-induced antigen retrieval, 5 μm sections of FFPE samples were tested for the presence of cIAP1 (Rabbit polyclonal IgG AF8181, R&D Systems, Wiesbaden, Germany), cIAP2 (Mouse IgG AF8171, R&D Systems), XIAP (Mouse IgG1, clone 48, BD Biosciences, Franklin Lake, NJ, USA), ML-IAP (Mouse IgG1, IMG-347A, Imegenex, Cambridge, UK), GFAP (EP672Y, Ventana Medical Systems, Illkirch, France), Ki67 (30-9, Ventana Medical Systems) and caspase-3 (C92605, BD Biosciences). A Benchmark Ventana autostainer (Ventana Medical Systems) was used for detection, and slides were simultaneously immunostained in order to avoid intermanipulation variability. For negative controls, irrelevant antibodies with identical isotypes were used. Slides were then scanned (Nanozoomer 2.0-HT, Hamamatsu Photonics SARL France, Massy, France) and images processed in NDP.view2 software (Hamamatsu).
Based on immunostaining results, ML-IAP positivity was determined as percentage of positive cells and cIAP1, cIAP2 and XIAP were quantified as positive or negative staining. The percentage of GFAP-, Ki67- or cleaved-caspase-3-positive cells was determined as previously described.^{26 (link)}

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).

A neuropathologist identified histologically representative tumor regions that were stained by hematoxylin and eosin. Tissue sections were cut at 4 µm and the IHC was performed using the Ventana Benchmark system (Ventana Medical System, Tucson, AZ, USA). As a pre-treatment step, tissues were subjected to heat-induced epitope retrieval with the Cell Conditioning 2 solution (Ventana, Tucson, AZ, USA), 24 min for Ki-67 (30-9) (Ventana, Tucson, AZ, USA), 32 min for p53 (DO-7) (Ventana, Tucson, AZ, USA) and IDH1 (R132H) (Dianova, Hamburg, Germany). The antibody concentrations were 2 µg/ml for Ki-67, 184 µg/ml for p53, and 4 µg/ml for IDH1. Two independent observers evaluated the stained slides. Proliferation index was evaluated using Ki-67 antibody staining and calculated by determining the percentage of immunopositive nuclei. A total of 100-500 nuclei were counted. The tumors were divided into two groups, less aggressive (<15 %) and more aggressive ≥15 %). The consensus for p53 was scored in four different categories: no immunoreactivity (0 %), faint (≤50 %), moderate (50–75 %), and strong (≥75 %) immunoreactivity. IDH1 was scored in two categories: (0–10 %) for negative immunoreactivity, and (≥10 %) for positive immunoreactivity.