All cells were fixed for 15 min with 4% paraformaldehyde in PBS (Sigma-Aldrich) and permeabilized with 0.5% Triton-X (Sigma-Aldrich) for >4 h at RT before staining. Hoechst (H1399; Thermo Fisher Scientific, Waltham, MA) was used at 5 μg/mL with 15 min incubation at RT to stain nuclei. Primary antibodies were applied overnight at 4°C in a blocking buffer of 5% donkey serum (Sigma-Aldrich) at the following concentrations: anti-laminin (L9393; Sigma-Alrich) 1:500; TRA-1-60 (MAB4360; EMD Millipore, Burlington, MA) 1:100; NANOG (AF1997; R&D Systems) 1:200; CD71 (334107; Biolegend) 1:100.
Secondary antibodies were obtained from Thermo Fisher Scientific and applied in a blocking buffer of 5% donkey serum for 1 h at RT at concentrations of 1:400 to 1:800. A Nikon Eclipse Ti epifluorescence microscope was used to acquire single 10 × images of each micropattern, and a Nikon AR1 confocal microscope was used to acquire 60 × stitched images of each micropattern using the z-plane closest to the micropatterned substrate for reprogramming studies. In brief, EPCs are identified as CD71+; Nanog−, IMs are indicated as CD71−; Nanog−, and iPSCs are indicated as CD71−; Nanog+.
Secondary antibodies were obtained from Thermo Fisher Scientific and applied in a blocking buffer of 5% donkey serum for 1 h at RT at concentrations of 1:400 to 1:800. A Nikon Eclipse Ti epifluorescence microscope was used to acquire single 10 × images of each micropattern, and a Nikon AR1 confocal microscope was used to acquire 60 × stitched images of each micropattern using the z-plane closest to the micropatterned substrate for reprogramming studies. In brief, EPCs are identified as CD71+; Nanog−, IMs are indicated as CD71−; Nanog−, and iPSCs are indicated as CD71−; Nanog+.