Ki 67 mib 1

In our IHC study, formalin-fixed, paraffin-embedded tissue sections obtained from surgical specimens were stained using appropriate antibodies specific for 4 markers: ER (1:100 dilution, clone 6F11; Novocastra, Newcastle upon Tyne, UK), PR (clone 16; Novocastra), HER2 (4B5 rabbit monoclonal antibody; Ventana Medical Systems, Tucson, US), and Ki-67 (MIB-1; Dako, Glostrup, Denmark). The HER2 status was defined as positive with a score of 3+ and negative with a score of 0 or 1+. Tumors with scores of 2+ were analyzed by fluorescent in situ hybridization following the manufacturer's protocol (PathVysion kit; Vysis, Downers Grove, US or HER2 inform; Ventana Medical Systems).

Immunohistochemical stainings were performed on FFPE tissue using fully automated protocols (DAKO Autostainer Link 48). Staining protocols with antibodies to MYC (clone Y69; Abcam), BCL‐2 (clone 124; DAKO), BCL‐6 (clone PG‐B6p; DAKO), Ki‐67 (MIB‐1; DAKO), MUM‐1(clone MUM1p; DAKO), CD10 (clone 56C6; DAKO) and p53 (DO‐7; DAKO) were performed. The estimation of positive staining for CD10, BCL‐6 and MUM‐1 was based on the Hans algorithm. Cut‐off values of 70% and 40% were used for BCL2 and MYC, respectively, as previously described.16

All tissues for immunohistochemistry were fixed in formalin and embedded in paraffin. Consecutive (semiserial) 4 μm sections of tissue samples were collected on poly-L-lysine coated slides. One section for each sample was stained with H and E. For immunohistochemical studies, the histologic sections were deparaffinized in xylene and rehydrated in graded alcohols up to water. Antigen retrieval was performed by microwaving the slides in 0.01 M citrate buffer (pH, 6). Endogenous peroxidase activity was quenched by treatment with 1% hydrogen peroxide for 20 minutes. Incubation with an appropriate protein-blocking solution was performed. Sections were subsequently incubated with primary antibodies: monoclonal mouse anti-PC2 (clone D-3) antibody (cat. no. sc-28331; dilution 1:100) (Santa Cruz Biotechnology) and Ki67 (MIB-1) (Dako). Detection was carried out using the Envision Plus Detection System kit, according to the manufacturer’s instructions (DakoCytomation), with 3,3΄-diaminobenzidine as a chromogen (which yielded brown reaction products). Sections were counterstained with Mayer hematoxylin solution, dehydrated, and mounted. To ensure antibody specificity, negative controls included the omission of primary antibodies and substitution with nonimmune serum. Positive human kidney tissue for PC2 was used as a positive control.