Ab86734

Tissues of tissue microarray (TMA) were sliced into sections with 9 µm thickness and mounted onto glass slices. Multiplex immunofluorescent staining was performed on TMAs using Opal 7‐Color IHC kits (Akoya Biosciences, Hopkinton, MA) according to the manufacturer's instructions. Briefly, sections were deparaffinized using xylene and progressively hydrated by ethanol ending with a distilled water wash. Microwave treatment was performed in AR6 buffer for antigen retrieval. Then slides were sequentially stained with the following primary antibodies and fluorescent dyes: anti‐CD31 (1:50, #ab9498, Abcam)/Opal‐520, Anti‐phospho‐FGFR4 (1:200, #PA5105531, Invitrogen)/Opal‐620, anti‐pan‐cytokeratin (1:100, #ab86734, Abcam)/Opal‐690. For each molecule being detected, slides were incubated with the specific primary  and secondary antibody, followed by Opal fluorophore solution and incubated for 10 min at room temperature. Afterward, microwave treatment was performed again to remove the specific primary antibody. Steps were repeated for subsequent primary antibodies to achieve multiplex IF staining. After antibody staining, slides were incubated in DAPI solution for 5 min at room temperature, washed several times, and then mounted with coverslips. Digital images of all cores of TMA were acquired using the Vectra Polaris Imaging System and analyzed by inForm software (Akoya Biosciences).

Primary endometrial stromal cells from women with adenomyosis were isolated according to a protocol with minor modifications (25 (link)). Briefly, endometrial tissues were homogenized and treated in sequential steps of pancreatin-0.05% trypsin enzymatic solution, collagenase 4 (0.1 U/ml), and DNase I (16 μg/ml) solution in Ca2+/Mg2+-free PBS (Gibco^® Thermo Fisher Scientific, Sweden) and incubated for 30 minutes at each step. Enzymatically digested cell suspensions were filtered through a 100 µM cell strainer to remove larger debris while collecting the flowthrough containing both the epithelial and stromal fractions. Later, these cell suspensions were adherent and expanded in vitro for two-three generations, frozen, and stored in liquid nitrogen for the following experiments. The isolated ESC was cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA) and 1% Penicillin-Streptomycin solutions (Gibco, USA). When the cells reached 80-90% confluency, the cells were passaged, and the medium was changed every 2-3 days. Cyto-immunofluorescent staining for vimentin (Abcam, ab16700) and pan-cytokeratin (Abcam, ab86734) was applied for confirmation of stromal cell phenotype.