Rabbit anti human cd31 primary antibody

Cells at day 1, 3 and 5 were fixed at room temperature for 30 min with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS, pH = 7.4), followed by blocking with 2% bovine aerum albumin for 30 min at room temperature. Samples were incubated overnight at 4 °C with 200 μL of mouse anti-human α-SMA primary antibody (Sigma) and rabbit anti-human CD31 primary antibody (Abcam, Cambridge, UK). After washing 3 times with PBS (pH = 7.4), samples were reacted with goat anti-mouse IgG secondary antibody (Alexa Fluor^® 488, Abcam) or anti-rabbit IgG secondary antibody (Alexa Fluor^® 594 Goat, Life Technology, Carlsbad, CA, USA) for 2 h at room temperature, then 1 mL (5 μg/mL) of 4',6-diamidino-2-phenylindole (DAPI) was added into each well to stain the nuclei at room temperature for 15 min, then washed with PBS (pH = 7.4) to remove excess DAPI dyestuff. Fluorescently-labelled cells were observed at 488 nm using an FV1000 confocal laser scanning microscope (Olympus).

A platelet-endothelial cell adhesion molecule (PECAM-1 or CD31) is an endothelial-specific adhesion protein and specific HUVEC marker. It was assessed by immunefluorescent staining. The effect of each β-TCP scaffold on expression of PECAM-1 was investigated at 7 and 14 days. Samples were rinsed and fixed in 4% paraformaldehyde solution (PFA) for 15 min at room temperature. The fixed cell/scaffold was placed in 3% bovine serum albumin (BSA) blocking buffer for 1 h. After incubating with rabbit anti-human CD31 primary antibody (1:50, Abcam) overnight at 4°C, the cell/scaffold was incubated in an anti-rabbit secondary antibody Alexa Fluor ®594 (1:1000; 2 μg ml⁻¹, Invitrogen) for 1 h at room temperature. Then the cell nucleus was counterstained with DAPI solution (5 μg ml⁻¹) for 3 min. The sample was mounted and imaged using confocal laser scanning microscopy (CLSM).