Eight hundred thousand B2m−/−Ciita−/− Cd47 tg miECs, miSMCs or miCMs in 1:1 diluted Matrigel (Corning) were injected into allogeneic BALB/c mice. Matrigel plugs were recovered after 1, 2, 3, 4, 5, 6 and 8 weeks and fixed in 4% paraformaldehyde in PBS with 1% glutaraldehyde for 24 h. Samples were dehydrated, embedded in paraffin and cut into sections of 5 µm thickness. For histopathology, sections were stained with hematoxylin and eosin (Carl Roth) and images taken with an inverted light microscope. Origin of cells was demonstrated with immunofluorescence staining. Sections were rehydrated, and underwent antigen retrieval and blocking. Samples were incubated with antibodies against luciferase (ab21176), SMA (ab21027, Abcam), VE-Cadherin (SC-6458) or α-sarcomeric actinin (EA-53, Abcam) and a corresponding secondary antibody conjugated with AF488 or AF555 (Invitrogen). Cell nuclei were counterstained with DAPI and images taken with a Leica SP5 laser confocal microscope (Leica).
For co-staining experiments of miECs and immune cells, primary antibodies were used against VE-Cadherin (SC-6458, Sigma) and CD3 (ab16669, Abcam), followed by the corresponding secondary antibody conjugated with AF488 or AF555 (Invitrogen).
For co-staining experiments of miECs and immune cells, primary antibodies were used against VE-Cadherin (SC-6458, Sigma) and CD3 (ab16669, Abcam), followed by the corresponding secondary antibody conjugated with AF488 or AF555 (Invitrogen).