Alexa fluor 488 fitc

Immunohistochemical stainings were performed to validate the scaffold´s functional integrity after re-endothelialization. In brief, after explantation the scaffold was fixed in 4% paraformaldehyde at room temperature for 2 h and subsequently embedded in paraffin. 5 μm sections were deparaffinized by dehydration and antigens retrieved as per primary antibodies manufacturer’s protocol. The primary antibody was incubated over night at 4 °C. The secondary antibody conjugated with fluorochrome was incubated for 1 h at room temperature. Washing steps were conducted subsequently after each step. All fluorescence imaging was performed on a Keyence BZ-9000 fluorescence microscope. The used antibodies were: CD31 (35 ng/ml; DAKO Cytomation), vWF (120 ng/ml; DAKO Cytomation), CK18 (5.4 mg/ml; DAKO Cytomation), Vimentin (300 ng/ml; Abcam), activated cleaved Caspase3 (2 µ/ml; Abcam), Ki67 (1:100; Abcam), CD90 (1:50; Abcam), Alexa Fluor 488 FITC, Alexa Fluor 647 (1:400; Invitrogen).