Ab150079

Cells were cultured on cover glasses in 24‐well cell culture plates, fixed with 4% PFA at room temperature for 10 min, and washed thrice with PBS. Rabbit anti‐E‐cadherin (Cell Signaling Technology, 3195, 1:200), mouse anti‐N‐cadherin (Invitrogen, 333900, 1:200), rabbit anti‐Nanog (Novus Biologicals, NB100‐58842, 1:300), and mouse anti‐Rex1 (made in‐house, 1:500) were added as the primary antibodies prepared with 0.3% Triton X‐100 and 10% goat serum in PBS at 4°C overnight. Then, cells were washed thrice with PBS. Goat anti‐rabbit Alexa 647 (Abcam, ab150079, 1:500) and goat anti‐mouse Alexa 568 (Invitrogen, A11004, 1:500) were added as the secondary antibodies prepared with PBS at room temperature for 1 h. Then, cells were washed thrice with PBS. Nuclei were stained with DAPI prepared in PBS at room temperature for 5 min. Image collecting was conducted with Zeiss LSM 800.

Microtissues were washed 3× times with PBS and then fixed overnight in 10% formalin at 4 °C. Following 3× washes with PBS, fixed microtissues were incubated for 4 h in permeabilization solution containing 0.2% Triton X-100 in PBS supplemented with 2% BSA, 320 mM sucrose and 6 mM magnesium chloride. Before immunostaining, microtissues were blocked in PBS containing 2% BSA for 2 h. Next, microtissues were stained with primary antibodies for cardiac troponin-t (cTnT) (Thermofisher MA5-12960, 1:200) and vimentin (Thermofisher MA5-16409; 1:200) to visualize cardiomyocytes and fibroblasts, respectively. To visualize sarcomeres, samples were stained with anti-sarcomeric alpha actinin antibody (abcam 137346; 1:200). To visualize gap junctions, samples were stained with connexin-43 (Thermofisher 71-0700; 1:200). Primary antibodies were diluted in PBS containing 3% BSA and hydrogels were stained at 4 °C for 72 h. Next, the secondary antibodies Alexa Fluor-488/594/647 IgG H&L (1:200; Abcam ab150077, ab150116, ab150079) were added at 4 °C for 72 h. Finally, samples were washed 3× in PBS followed by DAPI staining (1:2000; Invitrogen D1306, in PBS) for 30 min at room temperature. A Leica SP5 confocal was used to acquire z-stack images.