Antifade mounting reagent

The parietal peritoneum of abdominal wall was fixed with 4% paraformaldehyde (pH 7.4) and embedded in paraffin. Parietal peritoneum sections of 2 μm thickness were stained with Masson’s trichrome. The thickness of the parietal peritoneum, including the mesothelium and submesothelial interstitium, was measured with an Aperio ScanScope CS Slide Scanner system (Aperio Technologies, Vista, CA, USA); whole-slide digital images were captured with X20 objective. For immunofluorescence microscopy, 3 μm tissue sections were incubated with primary antibodies against cytokeratin (Thermo Fisher Scientific, Waltham, MA, USA) or α-SMA (Abcam) at 4 °C overnight after antigen retrieval in boiling citrate buffer (pH 6.0) for 10 minutes. Sections were then incubated for 1 hours with fluorescein-conjugated secondary antibodies (Alexa Fluor 488 and Alexa Fluor 594; Molecular Probes, Eugene, OR, USA). The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes), and slides were mounted with antifade mounting reagent (Molecular Probes). These sections were viewed under a confocal scanning laser microscope using LSM 5 EXCITER (Carl Zeiss).

Cells were fixed with methanol for 20 min and then permeabilized with
0.1% Triton X-100/PBS for 5 min. Samples were blocked with a
ready-to-use blocking solution (Histostain kit, Invitrogen) and incubated with
mouse-anti-FLAG/DDK and rabbit anti-ER-α antibody overnight at
4 °C. Cells were then stained with anti-rabbit IgG fluorescent
conjugate (Alexa Flour 488, Molecular Probes, Eugene, OR, USA), and nuclei were
counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Cells were mounted
with antifade mounting reagent (Molecular Probes), and examined by Leica confocal
fluorescence microscopy.

DAPI, Alexa Fluor 555-conjugated pan-CK (Cell Signaling Technology, USA), Alexa Fluor 555-conjugated EpCAM (Cell Signaling Technology, USA) and APC-conjugated CD45 (BD Biosciences, USA) antibodies were used to identify CTCs. The fluorescent dye-conjugated antibodies were added to cell smears and incubated for two hours at room temperature. After staining, slides were mounted in DAPI-containing anti-fade mounting reagent (Thermo Fisher Scientific, USA) and scanned using a Cytation 5 Cell Imaging Multi-Mode Reader (BioTeck, USA). All images were captured under the same conditions. We applied DAPI staining to label DNA for identifying nucleated cells, CK/EpCAM staining to label CTCs, and CD45 staining to label WBCs.

For in vitro differentiation, cells were seeded onto laminin-coated coverslips. For iNEP staining, 5.8 × 10⁴ cells were seeded, and 5 × 10⁵ cells were seeded to stain iMNP, iMN, and iMature MNs. After six days of cell culture, in the iNEP, iMNP, and iMN stages, immunofluorescence staining was performed. For the iMature MN stage, immunofluorescence staining was performed after 10 days of culture. The differentiated cells were fixed in 4% paraformaldehyde (PFA) for 30 min at RT. The cells were permeabilized with 0.1% Triton X-100 for 10 min at RT. Cells were blocked with PBS containing 2% BSA (PBA, Sigma-Aldrich) for 30 min at RT. Primary antibodies were diluted and incubated with 2% PBA for 2 h at RT. After washing, Alex Fluor-488 or -594 (Life Technologies) conjugated secondary antibodies diluted in 2% PBA were added and incubated at RT for 1 h. Cells were stained with 4′,6-diamidino-2-phenylindole (DAPI, Roche, Basel, Switzerland), washed, and mounted using an antifade mounting reagent (Thermo Fisher Scientific). The cells were observed under a fluorescence microscope (Carl Zeiss, Oberkochen, Germany) (x 200 magnification).

We performed immunofluorescence staining 48 h after the seeding of transfected HEK 293T cells into culture plates containing poly-lysine-coated glass coverslips. The samples were fixed in 4% paraformaldehyde for 30 min at room temperature (RT), and the coated glass coverslips were washed in Tris-buffered saline with 0.01% Tween 20. The cells were then permeabilized using 0.1% Triton X-100 for 10 min at RT and blocked with PBS containing 2% bovine serum albumin (BSA; Sigma-Aldrich, St Louis, MO, USA) (PBA) for 30 min at RT. To analyze the expression of PTHrP in transfection cells, double-labeling studies were performed; anti-PTHrP primary antibodies were added for 2 h at RT (1:200 in 2% PBA; Novusbio, Barton Lane, Abingdon, UK; #3H1-5G8) and Alexa Fluor 594-conjugated secondary antibodies were added for 1 h at RT (1:200 in 2% PBA; Life Technologies, Carlsbad, CA, USA). Cells were finally washed and mounted using the antifade mounting reagent (Thermo Fisher Scientific) and observed under a fluorescence microscope (Carl Zeiss); positive cells were defined as those with the co-localization of DAPI (blue), GFP (green), and PTHrP (red) signals (low-powered ×50 magnification).