Anti ki 67 clone 30 9

Systematic assessment of samples in 10% neutral buffered formalin blocks was performed using a tissue microarray (TMA). The approximate molecular subtypes were assessed by immunohistochemistry. The tumor markers anti-ER SP1, anti-PR 1E2, anti-HER2/neu (clone 4B5), and anti-Ki-67 (clone 30-9) were used (Roche Diagnostics). Tumors were rated ER/PR-positive when nuclear labeling was evident in more than 1% of tumor cells. A semi-quantitative score was used for HER2 staining. A score of 0, 1+, or 2+ was considered negative staining, and a score of 3+ was considered positive staining. Fluorescence in situ hybridization (FISH) was performed for samples with a 2+ score. The approximate molecular subtypes were clustered as a function of the immunohistochemical results as follows: luminal A (ER/PR-positive, HER2-negative, and Ki-67<14), luminal B/HER-negative (ER/PR-positive, HER2-negative, and Ki-67 ≥14), luminal B/HER-positive (ER/PR-positive and HER2-positive), HER2 (ER/PR-negative and HER2-positive), and triple negative (ER/PR-negative and HER2-negative). In the absence of TMA information, the primary immunohistochemical lamina was reviewed. Discrepancies were discussed until reaching consensus. To evaluate the analysis, luminal A and luminal B/HER-negative samples were grouped into the luminal/HER-negative group.

Immunohistochemistry (IHC) for Postn was performed using the Ventana Discovery automation system (Roche, Switzerland) according to the manufacturer's protocol. A rabbit polyclonal anti-Periostin antibody (ab14041, 1: 2,000, Abcam, UK) was used according to the manufacturer's instructions on 3-μm sections. Anti-Ki67 (clone 30-9, Roche) staining was also performed using the same system.