E cadherin

Immunohistochemical staining was performed on 10% formalin-fixed paraffin-embedded tissue blocks using a Discovery Auto-Stainer with automated protocols (Ventana Medical Systems, Inc.). The primary antibodies used were anti-E-cadherin (1:400; cat. no. 3195) and anti-vimentin (1:200; cat. no. 5741) from Cell Signaling Technology, Inc., and anti-ZEB1 (1:100; cat. no. ab203829) from Abcam, in addition to the Discovery Universal secondary antibody (cat. no. 760-4205). Two clinical pathologists (FS and YM) independently scored the staining intensity in ATC and non-cancerous tissues using a light microscope (magnification, ×40; Nikon Eclipse TS2; Nikon Corporation) for E-cadherin, vimentin and ZEB1 expression. Staining scores were recorded as follows: 0, negative staining in the cell membrane; 1, <10% of weak staining in the cell membrane; 2, >10% of weak or moderate staining in the cell membrane; and 3, >50% of strong staining in the cell membrane (15 (link)).

IHC were performed on 5-μm tumor sections in the Department of Pathology of the Centre Hospitalier Universitaire de Sherbrooke, Québec (CHUS) (Sherbrooke) using a standard streptavidin-biotin-peroxidase immunostaining procedure with a Ventana NexES autostainer and the solvent-resistant DAB Map detection kit (Ventana Medical Systems, Tucson, AZ) using ready-to-use solutions (Ki67 and E-cadherin) purchased from Dako, Burlington, Ontario, Canada.
Ki67-positive cells were manually counted in up to five × 400 light microscope representative fields per tumor (containing an average of 150 cells). Total counts were reported as total cell number per field. E-cadherin protein levels were quantified using the yellow channel of a cyan, magenta, yellow, key (CMYK) color model with pictures taken with a Super Coolscan 9000 scanner (Nikon, Tokyo, Japan) using Fiji software (Open Source) [13] (link), and quantification was performed using Image-Pro software (Media Cybernetics, Bethesda, MD). To avoid quantification of any nontumoral area (e.g., skin and fat), the xenograft sections were counterstained using hematoxylin and eosin in addition to staining the estrogen receptor, a positive marker of SKOV3 cells. Pictures with × 100 and × 400 magnifications were acquired using an Axioskop 2 phase-contrast microscope (Carl Zeiss, Thornwood, NY) and processed using Image-Pro software.