Cd146

Tissue sections or adherent cultured cells were incubated for one hour at room temperature in blocking solution (50 mmol/L Tris-HCl, pH 8.0, 0.1 mol/L NaCl, 0.1% Triton X-100, 3% NGS, 0.1% BSA) prior to overnight primary antibody incubation (4°C). Primary antibodies for immunofluorescence studies were: SOX2 (1:1000 dilution, Ab5603, Millipore), Ki67 (1:500 dilution, ab15580, Abcam), BrdU (1:500 dilution, ab6326, Abcam), NeuN (1:500 dilution, ab177487, Abcam), TUJ1 (1:200 dilution, T8660, Sigma-Aldrich), GFAP (1:500 dilution, Z0334, Agilent), MBP (1:300 dilution, SMI-99P, Covance), OLIG2 (1:200 dilution, AB9610, Millipore), OLIG2 (1:50 dilution, MABN50, Millipore), IBA1 (1:200 dilution, ab5076, Abcam), CD31 (1:100 dilution, ab24590, Abcam), ACTA2 (1:400 dilution, ab5694, Abcam), GFP (1:2000 dilution, ab13970, Abcam), CD31 (1:300 dilution, AF3628-SP, R&D), CD146 (1:100 dilution, 134701, BioLegend). We used immunofluorescence staining with Alexa Fluor 488, 555, or 647 (Life Technologies) and biotin-streptavidin-Alexa Fluor-conjugated secondary antibodies (Jackson ImmunoResearch), as well as horseradish peroxidase-based Vectastain ABC Kit (Vector Laboratories). Image acquisitions were performed using a Zeiss LSM880 confocal microscope and image editing done using ZEN, Photoshop or ImageJ.

Human ovarian isolated and cultured cells were processed for flow cytometry according to the Feisst et al. [18 (link)] procedure to determine the cell populations. Before staining, the frozen-thawed cells were washed and incubated in FBS culture medium at 37 °C, 5% CO₂ for 1 h. After washing with DPBS, frozen-thawed and cultured cells were stained with the following antibodies (Table 1): CD326 (324,233; BioLegend, the Netherlands), CD34 (343,615; BioLegend), CD31 (303,131; BioLegend), CD90 (328,113; BioLegend), CD73 (344,007; BioLegend), podoplanin (PDPN, 337005; BioLegend), CD146 (361,021; BioLegend), and CD105 (323,205; BioLegend) and dark-incubated at room temperature for 10 min.Table 1

CD markers used in cell sorting

CD markers	Cell types
CD326	Epithelial cells
CD34	Hematopoietic stem cells, endothelial progenitor cells
CD31	Endothelial cells
CD90	Stem cells, mesenchymal stem cells, fibroblasts
PDPN	Fibroblast, cancer-associated fibroblasts (CAFs)
CD73	Multiple cells, immunosuppressive enzyme
CD105	Endothelial cells, neoangiogenesis
CD146	Mesenchymal stem cells, angioblasts, endothelial cells, periendothelial cells

At least 10,000 events were analyzed by BD FACSCanto™ II Clinical Flow Cytometry System (BD Biosciences, Belgium). FlowJo software (BD Biosciences, USA) was used for data analysis.

The tissue sections were dried at RT for 20 minute and then blocked with 1% BSA PBS for 3×10 minutes, the sections were incubated with primary antibody at 4 °C overnight. The sections were washed with 1% BSA PBS buffer and then incubated with the secondary antibody conjugated with Alexa-dye for 1 hour at RT in dark room. The sections were washed with 1% BSA PBS buffer and stained with 0.1% DAPI for 5 minutes at RT in dark room. The sections were then washed and mounted in anti-fade mounting media (Vector Laboratories). The stained slides were observed using an Olympus IX81 microscope or scanned using an iCys Research imaging cytometer (CompuCyte).
The following antibodies were used in this study: Col2 (1:100, Cat. ab34712, Abcam), osteocalcin (1:200, Cat. Ab14173, Abcam), Kitl (1:100, Cat. sc-13126, Santa Cruz), CXCL12 (1:100, Cat. sc-6193, Santa Cruz), α-SMA (1:100, Cat. ab5694, Abcam), CD146 (1:100, Cat. 134702, Biolegend), CD140a (1:100, Cat. 135902, Biolegend), Sca-1 (1:100, Cat. 108102, Biolegend), CD31 (1:150, Cat. 5550274, BD). The secondary antibodies conjugated with Alexa fluor 488 and Alexa fluor 555 were from Invitrogen. In study with mouse primary antibody, the M.O.M. immunodetection kit (Cat. BMK-2202, Vector Laboratories) was used to eliminate the endogenous mouse immunoglobulins background in the tissue.