Prior to immunofluorescent stainings, the hydrogel constructs were
permeabilized with 0.2% triton-X in PBS for 30 min and blocked in 5% BSA/PBS for
30 min. Capillarylike structures in the hydrogels were investigated by CD31
staining (5.1 μg mL−1, M0823, Dako), secondary sheep antimouse
biotinylated antibody (1:200, RPN1001v1, GE Healthcare), and tertiary
streptavidin Alexa Fluor 488 conjugate (5.0 μg mL−1, S32354,
Invitrogen). In vasculogenic cocultures, ECFCs with the GFP label were not
stained for CD31. The endothelial phenotype was confirmed by a rabbit
antivascular endothelial cadherin antibody (VE-cad, 1:250, D87F2, Cell
Signalling Technology) which was combined with a secondary donkey-antirabbit
Alexa 647 antibody (5 μg mL−1, ab150075, Abcam). Stabilizing cells of
the capillary-like structures were identified by a mouse monoclonal
Cy3-conjugated aSMA antibody (1:300 μg mL−1, Clone 1A4, C6198 Sigma
Aldrich). Furthermore, 4,6-diamidino-2-phenylindole (DAPI, 100 ng
mL−1, Sigma) was used to stain cell nuclei. The hydrogels were
imaged with a confocal microscope (SP8x Leica, DMi8, Leica).
permeabilized with 0.2% triton-X in PBS for 30 min and blocked in 5% BSA/PBS for
30 min. Capillarylike structures in the hydrogels were investigated by CD31
staining (5.1 μg mL−1, M0823, Dako), secondary sheep antimouse
biotinylated antibody (1:200, RPN1001v1, GE Healthcare), and tertiary
streptavidin Alexa Fluor 488 conjugate (5.0 μg mL−1, S32354,
Invitrogen). In vasculogenic cocultures, ECFCs with the GFP label were not
stained for CD31. The endothelial phenotype was confirmed by a rabbit
antivascular endothelial cadherin antibody (VE-cad, 1:250, D87F2, Cell
Signalling Technology) which was combined with a secondary donkey-antirabbit
Alexa 647 antibody (5 μg mL−1, ab150075, Abcam). Stabilizing cells of
the capillary-like structures were identified by a mouse monoclonal
Cy3-conjugated aSMA antibody (1:300 μg mL−1, Clone 1A4, C6198 Sigma
Aldrich). Furthermore, 4,6-diamidino-2-phenylindole (DAPI, 100 ng
mL−1, Sigma) was used to stain cell nuclei. The hydrogels were
imaged with a confocal microscope (SP8x Leica, DMi8, Leica).