Alexa fluor 488 or 594 conjugated goat antirabbit

Cell surface antigens on cells were evaluated with FACS. The cells were dissociated with 0.05% trypsin/EDTA (Highclone), washed with PBS, fixed with 4% paraformaldehyde, permeabilized with Triton X‐100, and blocked with a BSA mixture. The samples were then stained with antibodies against human octamer‐binding transcription factor 4 (OCT4; R&D systems, 1:200), stage specific embryonic antigen 1 (SSEA1; R&D systems, 1:200), cluster of differentiation 31 (CD31‐FITC; Miltenyi Biotec, 1:100), CD34 (CD34‐PerCP, BioLegend, 1:100), human leukocyte antigen ‐ DR isotype (HLA‐DR, HLA‐DR‐APC, BioLegend, 1:100), CD73 (CD73‐PE, BioLegend, 1:100), CD90 (CD90‐APC, BioLegend, 1:200), CD105 (CD105‐FITC, BioLegend, 1:150), integrin α5 (Santa Cruz, 1:200), integrin α11 (Abcam, 1:200), integrin β1(Abcam, 1:200), and integrin β5 (BioLegend, 1:200) for 30 min or 1 h at 4 °C. Samples were subsequently stained with fluorescently labeled secondary antibodies (Alexa Fluor‐488 or 594 conjugated goat antirabbit (Abcam, 1:400), Alexa Fluor‐488 or 594 conjugated goat antimouse (Abcam, 1:400)) for 30 min at 4 °C. The corresponding mouse/rabbit isotype antibodies (Abcam, 1:200) were used as controls. Cell immunotypes were determined with the Accuri C6 flow cytometer (BD Biosciences) and the percentage of expressed cell surface antigens was calculated for 10 000 gated‐cell events.

Samples were fixed with 4% paraformaldehyde (Sigma), permeabilized with Triton X‐100 (Sigma), and blocked with a mixture of bovine serum albumin (BSA, Sigma) and either goat or donkey serum, depending on the species of secondary antibody used. Samples were incubated with primary antibodies (Antibeta Actin (Santa Cruz, 1:200), Anti‐Vinculin (Millipore Sigma, 1:200), Anti‐FAK (Millipore Sigma, 1:200), and Anti‐Integrin α5β1 (Millipore Sigma, 1:200)) overnight at 4 °C and washed. The samples were subsequently stained with fluorescently labeled secondary antibodies (Alexa Fluor‐488 or 594 conjugated goat antirabbit (Abcam, 1:400), Alexa Fluor‐488 or 594 conjugated goat antimouse (Abcam, 1:400)) for 30 min at RT. 4′,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI, Sigma) was applied as a nuclear counterstain for 5 min at RT. The samples were washed and mounted for imaging with a confocal microscope (LSM 710, Zeiss) and fluorescence microscope (IX71 inverted microscope, Olympus). The relative surface area of coverage for stains was quantified with the ImageJ software (NIH, version 1.25p).