Ep1628y

Six to eight week-old immunodeficient SCID-beige mice were used for transplantation of WT hiPSC or B2M^−/−CIITA^−/− CD47 tg hiPSCs. Here 1 × 10⁷cells were resuspended in 100 µl saline solution and injected into the right thigh muscle of the mice. Teratomas were recovered, fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin and cut into sections of 5 µm thickness. For histopathology, sections were rehydrated and stained with hematoxylin and eosin. Images were taken with an inverted light microscope. For immunofluorescence, slides underwent heat-induced antigen retrieval in a steamer with Dako antigen-retrieval solution (Dako), followed by antigen blocking with Image-iT FX signal enhancer solution (Invitrogen). Tissue sections were incubated with a primary antibody against brachyury (Ab20680, Abcam), followed by a goat anti-rabbit IgG secondary antibody conjugated with Alexa Fluor 555 (Invitrogen). Subsequently, sections were incubated with primary antibodies against cytokeratin 8 (EP1628Y, Abcam) and GFAP (GA5, Cell Signaling) conjugated with AF488 or AF647, respectively. DAPI was used to counterstain cell nuclei and images were acquired with a Leica SP5 laser confocal microscope (Leica).