Immunofluorescence Imaging of Endometrial Tumors

All patient samples were collected as described previously [52 (link)] at MDACC and processed at Rice University under approval of Institutional Review Boards from each institution. Endometrial tumor grade and histotype can be found in Supplementary Table 2. Cells were grown in Nunc Lab-Tek II chambered glass slides (Sigma-Aldrich) to ~80% confluence. Cells and tissues were fixed with 4% (w/v) paraformaldehyde (Fisher Scientific) in PBS for 10 min, permeabilized with 0.25% (v/v) triton X-100 in PBS for 2 min and then blocked with 1% (w/v) BSA in PBS for 1 hr. This was followed by incubation in a 1:200 dilution of primary antibody in blocking buffer for 1 hr and then a 1:400 dilutions of secondary antibody in blocking buffer. Fixing, permeabilization and antibody incubation steps were followed by a 3 × 5 min wash with PBS. Slides then were sealed with ProLong Gold Antifade with DAPI (Life Technologies) and a coverslip. All slides were imaged with a Nikon A1-Rsi confocal microscope (Nikon, Tokyo, Japan) or a Zeiss LSM 710 confocal (Carl Zeiss AG, Jena, Germany).