Anti ki67 30 9 rabbit monoclonal primary antibody

Ki67 immunohistochemistry data was obtained from central review blinded from clinical-pathological and outcome data. Ki67 was assessed by immunohistochemistry using anti-Ki67 (30-9) rabbit monoclonal primary antibody (Ventana Medical System). In all samples, Ki67 interpretation criteria were done according to the latest international recommendations [28 (link)].

Hematoxylin and eosin (H&E)‐stained sections were examined for morphologic features. Epidermal proliferation and differentiation were assessed by immunohistochemical (IHC) staining for Ki‐67‐positive nuclei using anti‐Ki‐67 (30‐9) rabbit monoclonal primary antibody (Ventana Roche, Tucson, Arizona). To analyze the Ki‐67‐positive cells of each section, a line of 1‐mm length following the stratum basale was drawn after choosing a representative region. All positive cells above and under the line were counted by two independent observers (LL and KP) and expressed as “positive cells per millimeter length of basement membrane.”

Sections (3 μm-thick) were cut from formalin-fixed paraffin-embedded (FFPE) tumor tissue blocks and stained with H&E or processed for immunohistochemistry (IHC) analysis. The latter was performed using an automated platform (Ventana BenchMark AutoStainer, Ventana Medical Systems, Tucson, AZ, USA) with the following primary antibodies: anti-Ki-67 (30-9) rabbit monoclonal primary antibody (cat:790-4286, Ventana Medical Systems); cleaved Caspase-3 (Asp175) polyclonal rabbit antibody (cat:9661, Cell Signaling, Danvers, MA, USA). Antigen retrieval was performed using ULTRA Cell Conditioning Solution (ULTRA CC1) antigen retrieval buffer (pH 8.5, Ventana Medical Systems) for all sections and the Ultraview detection system (Roche Diagnostic, Milano, Italy) was used for all analysis. Appropriate positive and negative controls were included for each immunohistochemical analysis. Image acquisition was performed with Hamamatsu NanoZoomer S210 Digital Slide Scanner (Iwata-City, Japan). The number of mitotic cells was assessed by counting number of mitoses per 10 high power fields (mitosis/10HPF) on standard Hematoxylin and Eosin (H&E) stained slides (40 × magnification). Only viable cells were considered in each HPF examined within the section.