Anti rabbit cd31 primary antibody

To evaluate differentiation of stem cells and endothelial cells, they were fixed with 10% NBF for 1 h, blocked with 2% bovine serum albumin (BSA; Sigma‐Aldrich) for 2 h, and permeabilized with 2% Triton X‐100 for 2 h. The samples were incubated with an anti‐mouse osteopontin (OPN) primary antibody (5 μg/ml; Invitrogen, USA) and an anti‐rabbit CD31 primary antibody (5 μg/ml; Invitrogen, USA) overnight at 4°C. The primary antibody‐treated samples were stained with Alexa Fluor 488‐conjugated anti‐mouse secondary antibody (1:50 in DPBS; Invitrogen, USA) or Alexa Fluor 594‐conjugated anti‐rabbit secondary antibody (1:50 in DPBS; Invitrogen, USA) for 1 h, and counterstained with DAPI (5 μM in DPBS). The confocal microscope was used to visualize the stained cells. OPN‐ and CD31‐positive areas were quantified using ImageJ software. All values are expressed as means ± SD (n = 4).

To evaluate the differentiation of hASCs and ECs, the cells were treated with NBF (10%; fixation) for 1 h, bovine serum albumin (BSA; Sigma-Aldrich) (2%; blocking) for 2 h, and Triton X-100 (2%; permeabilization) for 2 h. The cells were then treated with an anti-mouse Ve-cadherin primary antibody (5 μg/mL; Invitrogen), an anti-rabbit CD31 primary antibody (5 μg/mL; Invitrogen), and an anti-mouse osteopontin (OPN) primary antibody (5 μg/mL; Invitrogen) overnight at 4 °C, followed by staining with the Alexa Fluor 488-conjugated anti-mouse secondary antibody (1:50 in DPBS; Invitrogen) and Alexa Fluor 594-conjugated anti-rabbit secondary antibody (1:50 in DPBS; Invitrogen) for 1 h according to the host species of the primary antibody. The cells were counterstained with DAPI (5 μM in DPBS) to visualize nuclei. The stained cells were observed under a confocal microscope. Quantification of the OPN⁺ and CD31⁺ areas was performed using ImageJ software. All values are reported as mean ± SD (n = 4).