Ab87280

FFPE sections from each xenografted tumor were stained using antibodies against ZEB1, E-cadherin and vimentin. The IHC conditions for E-cadherin (monoclonal, Ventana) and vimentin (monoclonal, Dako) were previously optimized by the Memorial Sloan Kettering Cancer Center Pathology Core Laboratory. For ZEB1 staining, epitope retrieval was performed using heat by steaming with Citrate buffer (Dako Target Retrieval Solution, #S1699) at pH 6 for 20 minutes. Sections were incubated overnight with the primary antibody at 4 °C (Anti-AREB6, Abcam ab87280, 1:200). Bound antibodies were then detected using Envision + HRP-labeled polymer anti-rabbit for 30 minutes at room temperature (Dako, K4003). For tumors with complete loss of protein expression, staining of blood vessels and stromal cells served as positive internal controls. All slides were reviewed for morphology and IHC staining by an expert gynecologic pathologist.

Immunohistochemical analysis of paraffin-embedded sections was performed using the Envision Kit (Dako) following the manufacturer’s protocols. Sections were boiled in retrieval solutions to expose antigens. We applied rabbit polyclonal Ab against ZEB1 (ab87280; Abcam), rabbit polyclonal Ab against VEGFA (19003-1-AP; Proteintech), rabbit polyclonal Ab against CD31 (11265-1-AP; Proteintech), and control primary Abs to the sections at 1:100 dilution. Slides were counterstained with hematoxylin, dehydrated, and mounted. Immunostaining was independently evaluated by 2 pathologists.
To calculate MVD, the 5 most-vascularized areas of the tumor were selected and mean values obtained by counting vessels. A single microvessel was defined as a discrete cluster of cells positive for CD31 staining, with no requirement for the presence of a lumen.

Immunohistochemistry was carried out following the manufacturer’s recommendations. Tissue sections were embedded in paraffin and deparaffinized with xylene for 15 min, fixed with 100% ethanol for 10 min, and then rehydrated. The prepared slices were treated with methanol solution containing 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. Sections were washed twice with PBS and then incubated with antibodies against Snail, E-cadherin, Slug, ZEB1, Twist, Vimentin and Survivin overnight (dilutions 1:200, 1:100, 1:100, 1:50, 1:75, 1:200, and 1:100) at 4°C. All sections were subjected to a heat-induced antigen retrieval process. Sections were again washed with PBS twice and then incubated with the corresponding secondary antibody under ambient conditions for 30 min. Then, after color development with 3-amino9-ethylcarbazole for 15 min, the slides were counterstained with hematoxylin. Finally, an optical microscope was used to observe the slides. Negative and positive controls were used to optimize staining. EMT inducers (Snail, Slug, ZEB1, and Twist) and EMT markers (E-cadherin, Vimentin, and Survivin) were also used. Rabbit anti-human polyclonal antibodies against Snail (ab180714), E-cadherin (ab15148), Slug (ab180714), ZEB1 (ab87280), Twist (ab49254), Vimentin (ab45939), and Survivin (ab469) were purchased from Abcam (Cambridge, United Kingdom).