Ab80922

Extracted tumours were fixed in 10% buffered formalin (Sigma) for 24-48 h at room temperature and then stored in 70% ethanol at 4°C. Samples were then paraffin-embedded, sectioned at 4 μm thickness and stained with haematoxylin and eosin (H&E). Ki67 (NB110-90592, Novus), TUNEL (homemade by the Pathology Research Program Laboratory), ER (ab80922, Abcam), PR (NCL-PR-312, Leica), HER2 (RM9103, Thermo Fisher Scientific), CK5 (NCL-LCK5, Leica), EGFR (28-0005, Invitrogen), p63 (VP-P960, Vector) and SMA (M0851, Dako) were used to further analyse tumour sections. All immunohistochemistry was performed as a service by the Pathology Research Program Laboratory (University Health Network, Toronto, Canada).

Immunohistochemical staining of FFPE slides was performed by ARUP Laboratories using an automated immunostainer. Briefly, 5 μm tissue sections were obtained using a standard microtome and de-paraffinized with the EZ Prep solution. The slides were then treated with Cell Conditioning 1 (CC1), pH 8.5 for 68 min at 95 °C. The primary antibody (#ab80922, Abcam) was applied for 1 h at a dilution of 1:100 at 35 °C. Following removal of the primary antibody, the secondary antibody (#B8895, Sigma-Aldrich) was applied for 1 h at a dilution of 1:100 at 37 °C. Slides were then exposed to the IView DAB Map detection kit (Ventana) and counterstained with hematoxylin for 8 min. After dehydration in graded alcohol, coverslips were put on and slides were read by board-certified pathologists. Hematoxylin and eosin (H&E) staining was performed on 5 μm tissue sections.

Immunohistochemistry (IHC) was performed on formalin-fixed paraffin-embedded 5 μm sections from endometrial biopsies. Expression of PR was visualized using anti-PR (LabVision RM-9102-S0, Thermo Fisher Scientific) at 1:250; ER was evaluated using anti-ER-alpha (Abcam ab80922, 1:250), and Ki67 was visualized using anti-Ki67 (Dako, MIB-1, 1:100). Biotinylated goat anti-rabbit or biotinylated rabbit anti-mouse (Jackson ImmunoResearch, 1:1000) followed by streptavidin-conjugated horseradish peroxidase (HRP, Jackson ImmunoResearch, 1:1000) was used for DAB staining.