Immunofluorescence Analysis of Endometrial Markers

For immunofluorescence (IF) analysis, tissue sections were processed as previously described [52 (link),80 (link)] and incubated with the following primary antibodies: polyclonal rabbit anti-FOXL2 (homemade [81 (link)], 1/300), anti-α-SMA (Abcam ab124964, 1/500, Cambridge, UK), anti-Cx43 (Sigma Aldrich C6219, 1/400, St. Louis, MO, USA), and monoclonal mouse anti-PCNA (Sigma Aldrich P8825, 1/500), anti-pan-cytokeratin (Thermo Fisher MA5-13156, 1/400, Waltham, MA, USA). Sections were then incubated with donkey or goat secondary antibodies conjugated with Alexa Fluor 555, 488, or 649 (Thermo Fisher, 1/1000), followed by Hoechst staining. IF images were captured with a Zeiss AxioImager apotome microscope (Carl Zeiss Microscopy, Jena, Germany) at the IGH Imaging facility (BioCampus) and processed with the OMERO software (OMERO web 5.5.1., University of Dundee and Open Microscopy Environment, Dundee, UK). The intensity of α-SMA staining was determined in regions of interest (ROIs) (n = 4–12 ROIs per section) of a constant surface (30 mm²) at the endometrial stroma-junctional zone (2–3 sections of each uterus, n = 4–7 per group). Cx43 staining intensity was determined in ROIs (n = 4–10 ROIs per section, n = 3 per group) in the endometrial stroma (1500 μm²), luminal epithelium (1000 μm²), and glandular epithelium (700 μm²) (OMERO software). Data were analyzed with GraphPrism 7.