Lsm zen 2010

For IF staining of cells, around 8,000–10,000 cells were transferred onto each 3-aminopropylterithoxy saline (AAS, Sigma-Aldrich)-coated slide using the Shandon Cytospin Centrifuge (Thermos Electron, USA, Cambridge, UK) at the speed of 1,700 rpm for 10 min. Cell fixation was accomplished by 4% paraformaldehyde (PFA) for another 10 min at room temperature. For tissues, paraffin-embedded endometrial tissues were attached to the AAS-coated slides, followed by dewax, antigen retrieval, denaturation, and quenching procedures. To visualize protein expression in the nucleus, cells were permeabilized with 0.1% Triton X-100 for 15 min. Primary antibodies (Supplementary Table S3) were added and incubated overnight at 4°. On the following day, corresponding secondary antibodies were then added at room temperature for 1 h. Cell nuclei were stained with DAPI (Thermo Fisher Scientific), and the slides were mounted with a fluorescence mounting medium (Dako, Glostrup, Denmark). The fluorescent images were captured by a Carl Zeiss LSM 780 inverted confocal microscope and analyzed by Zeiss LSM Zen 2010 software (Carl Zeiss, Munich, Germany) at the Centre for PanorOmic Sciences (CPOS), the University of Hong Kong.

Double immunofluorescent staining was performed to evaluate the phenotypic of CD140b⁺CD146⁺ cells. Some of the cells after magnetic microbeading against CD140b and CD146 were plated at low seeding density of 10–30 cells/cm² on fibronectin-coated 12-well plates and culture for 3–4 days. Cells were fixed with 4% paraformaldehyde for 20 min. Permeabilization was performed using 0.1% Triton-X 100 for 10 min and blocked with 2% BSA for 30 min. Cells were incubated with primary antibodies; anti-human CD140b (1:200; R&D Systems) and anti-human CD146 (1:100; Abcam, Cambridge, UK) antibodies at 4 °C overnight. The following day, cells were incubated with the secondary antibodies: donkey anti-mouse antibodies conjugated with Alexa Fluor 564 (1:200; ThermoFisher Scientific) and goat anti-rabbit antibodies conjugated with Alexa Fluor 488 (1:200; ThermoFisher Scientific). The cell nuclei were detected by DAPI (ThermoFisher Scientific). All washing steps were performed with PBS and conducted at room temperature unless specified. Images were captured using a Carl Zeiss LSM inverted confocal microscope and a Zeiss LSM Zen 2010 software (Carl Zeiss, Munich, Germany) at the Centre for PanorOmics Sciences (CPOS) Imaging and Flow Cytometry Core, The University Of Hong Kong.

For immunofluorescence staining, ~8000 cells were cytospun at 12,000 rpm for 10 min and fixed with 4% paraformaldehyde for 10 min. Permeabilization was conducted with 0.1% Triton-X 100 for 10 min, followed by blocking with 5% BSA for 30 min. Primary antibodies (Table S2) or isotype-matched control antibodies were incubated overnight at 4 °C. The next day, the corresponding secondary antibody (Table S3) was added and incubated for 1 h. The cell nuclei were detected by staining with DAPI (Thermo Scientific) and mounted with mounting medium (Dako). The slides were washed with PBST between steps and all the steps were conducted at room temperature unless specified. Multi-spectrum fluorescence images were captured using a LSM 710 inverted confocal microscope and a LSM ZEN 2010 software (Carl Zeiss, Munich, Germany) at the CPOS, The University of Hong Kong. The total cell number and number of triple positive (CD140b⁺CD146⁺NICD⁺) cells were counted. At least 500 cells were counted from each sample.