Background snipper

Formalin-fixed paraffin-embedded tumor sections (4 μm) were deparafinized and rehydrated in a series of decreasing concentrations of ethanol. Antigen retrieval was performed at 110°C for 10 minutes using 10 mM sodium citrate buffer pH 6.0 (Decloaking Chamber, Biocare Medical). The samples were then incubated in 3% H₂O₂ (10 minutes). Tissue sections were blocked using Background Snipper (Biocare Medical) and incubated with CD73 (1:200, Cell Signaling (D7F9A, #13160) overnight at 4°C. A universal Dako-Labeled Streptavidin-Biotin2 System, Horseradish Peroxidase (LSAB2) System (#K0675), and Dako DAB (#K0673) were used to develop the staining. Tumor images (9–36 per sample, 10X magnification) were captured using a BX41 Olympus microscope. CD73 staining was quantified using QuPath software (57 ). Classifiers identifying tumor and stromal cells were built by manual selection of training data, then analyzed. Thresholds were set at 0.15 and DAB OD mean measured.

EC cell lines, grown on glass coverslips, and mouse uterine cryosections were fixed with 4% paraformaldehyde, incubated with 0.1% Triton X-100, and blocked with Background Snipper (Biocare Medical). Coverslips were labeled with CD73 (1:200, Cell Sciences (4G4, #HM2215) and/or Alexa Fluor 488 phalloidin (F-actin, Invitrogen). Cryosections were doubled-labeled with E-cadherin and phalloidin. Coverslips and cryosections were incubated overnight at 4°C, followed by appropriate secondary antibody. Images were captured using a BX41 Olympus microscope and analyzed using CellSens Dimension Software (Olympus). F-actin intensity in endometrial epithelial cells (glandular and luminal) was measured by manual selection (region of interest, ROI) and Adaptive Threshold. ROI selections were guided by E-cadherin positivity and H&E serial sections (data not shown).