Ki 67 clone 30 9

Immediately after samples were removed from culture, they were placed in 4% paraformaldehyde for fixation for at least one week and then embedded in paraffin. Once all samples were in paraffin, they were sectioned along the sagittal plane and stained on the same day. Tissue were stained with hematoxylin and eosin for morphological analysis, α-smooth muscle actin α-SMA (clone 14A, Cell Marque) for myofibroblast formation and intercapsular adhesion^{28 (link)–30 (link)}, Ki-67 (clone 30–9, Ventana) for proliferation^{31 (link)} and vimentin (clone V9, Ventana) a marker of both undifferentiated lens epithelium as well as differentiating fiber cells^{32 (link)}. All immunostainings were done with the BenchMark® ULTRA device (Ventana Medical Systems, Inc.). We also included negative immunostain controls to support the validity of our stains and identify possible experimental artefacts or background noise. These controls were processed in the same manner with the automated staining device but without the primary antibodies. Capsular adhesion, cellular morphology and the degree of staining of α-SMA, Ki-67 and vimentin were studied at the equatorial region of the capsule and at the center of the posterior capsule.

Sections were cut at 3 to 4μm intervals and immuno-stained using the monoclonal antibody Ki-67 (clone 30-9, pre-diluted, Ventana-Roche, Arizona, USA). Immunohistochemical stains were subsequently performed using Bench Mark GX with the iView DAB Detection kit (Ventana-Roche, Arizona, USA).

One representative formalin-fixed paraffin-embedded tissue block from each case (31 low-grade phyllodes tumor, 30 fibroadenoma, and 10 high-grade phyllodes tumor) was selected to generate 4 μm thick unstained slides for immunohistochemical staining for the following antibodies: Ki-67 (clone 30-9, prediluted, Ventana), p53 (clone Bp53-11, prediluted, Ventana), β-catenin (clone 14, prediluted, Ventana), and E-cadherin (clone EP700Y, prediluted, Ventana). The immunohistochemistry staining was performed on Ventana Benchmark automated immunostainer (Ventana Medical Systems, Inc., Tucson, AZ) according to standard protocols with appropriate positive and negative controls. The immunohistochemistry staining results were interpreted individually by two pathologists (C.Y. and L.Z.) blinded of the diagnosis. The percentage of cells positive was visually estimated and recorded and the average of the two results were used for analysis. Only nuclear staining for Ki-67, p53, and β-catenin in the stromal tumor cells were considered as positive. In contrast, cytoplasmic and membranous staining in the stromal tumor cells for E-cadherin was interpreted as positive. The percentage of staining was estimated relative to total number of stromal cells.