Ma5 12960

Immunocytochemical staining was performed to confirm the presence of cardiomyocytes and vascular networks in the cardiac tissue constructs. The cells were fixed with 70% ethanol for 20 min on day 9 after cardiomyocyte seeding. The cells were stained with cardiomyocyte-specific monoclonal mouse anti-troponin T antibody (1:100, Invitrogen, MA5-12960, lot UF2730196) and endothelial cell–specific polyclonal rabbit anti–von Willebrand factor (1:100, Dako, A0082, lot 20067357) at +4°C overnight. Secondary antibodies anti-mouse IgG Alexa Fluor 488 (Invitrogen, A21202, lot 2018296) and anti-rabbit IgG Alexa Fluor 594 (Invitrogen, A21207, lot 2066086) were incubated for 45 min at RT. The cells were imaged using an Olympus IX51 inverted fluorescence microscope using a ×10 objective. The images were prepared with Photoshop CC (Adobe).

Differentiated ES cells were plated on glass slides at an appropriate density. Then they were fixed with 4% paraformaldehyde (pH 7.4) for 10 min, permeabilized with 0.1% Triton dissolved in PBS for 10 min at RT, and incubated overnight in blocking solution containing antibody against NKX2-5 (sc-376565, Santa Cruz) or TNNT2 (MA5-12960, Invitrogen). A second antibody conjugated with TRITC or FITC were used for immunostaining. All immunofluorescence images were captured by a laser scanning confocal microscope system (LSM710, Zeiss).

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.

Jurkat cells, DND41, MOLT4, purified leukemia cells, and tissue sections were fixed by 4% paraformaldehyde (PFA). Anti‐γ‐H₂AX (rabbit, 1:100, 7631S, Cell Signaling Technology) or anti‐cTnT (MA5‐12960, ThermoFisher) were stained, followed by a donkey anti‐rabbit AF488 (1:500, A21206, Thermo Scientific) secondary antibody staining. The apoptotic cardiomyocytes were detected by TUNEL assay according to the manufacturer's instruction (C1088, Beyotime). For high‐resolution three‐dimensional images, z‐stack collected images from Nikon C2plus were analyzed with ImageJ (National Institutes of Health).