Anti ssea 3

Immunofluorescence staining for pluripotency of iPSCs was performed by using the following primary antibodies: anti‐NANOG (Abcam), anti‐OCT3/4 (Santa Cruz), anti–SSEA 3 (Millipore), anti–SSEA 4 (Millipore), anti–Tra‐1‐60 (Millipore), and anti–Tra‐1‐81 (Millipore). In immunofluorescence staining for cardiac markers, monoclonal anti–α‐actinin (Sigma), monoclonal anti‐cTnT (Thermo Scientific), polyclonal anti‐cTnT (Santa Cruz), monoclonal anti–myosin light chain (MLC)2a (Synaptic Systems), polyclonal anti‐MLC2v (ProteinTech Group), anti‐ANP (Santa Cruz), anti–cMyBP‐C (Santa Cruz), polyclonal anti–cMyBP‐C motif (supplied by C. Witt University of Heidelberg, Heidelberg, Germany), and anti–nuclear factor of activated T cells (NFAT)c4 (Santa Cruz) were used. The isotype‐specific secondary antibodies, Alexa Fluor 488 chicken anti‐rabbit IgG, Alexa Fluor 594 goat anti‐mouse IgG₁, Alexa Fluor 488 goat anti‐rat IgM, Alexa Fluor 594 goat anti‐mouse IgM, Alexa Fluor 488 goat anti‐mouse IgG, Alexa Fluor 594 goat anti‐mouse IgG_2b, Alexa Fluor 594 chicken anti‐goat IgG, and Alexa Fluor 555 goat anti‐rabbit IgG, were all obtained from Invitrogen. The tested drugs included endothelin‐1 (1.0, 10, 100, or 1000 nmol/L), angiotensin II (100 nmol/L), insulin‐like growth factor 1 (100 nmol/L), phenylephrine (0.05 mmol/L), BQ‐123 (250 nmol/L), and BQ‐788 (100 nmol/L) (all from Sigma).

Colonies of undifferentiated human iPSCs plated on MEF feeder cells and cardiomyocytes plated on fibronectin-coated dishes were fixed with 4% paraformaldehyde (MUTO Pure Chemicals, Tokyo, Japan) for 30 min at 4 °C. After fixation, cells were permeabilized with 1% Triton X-100 and blocked with ImmunoBlock (DS Pharma Biomedical, Osaka, Japan). Specimens were incubated at 4 °C overnight with each of the following primary antibodies: anti-OCT3/4 (Santa Cruz, CA, USA), anti-NANOG (Abcam, Camb, UK), anti-SSEA3 (Millipore, MA, USA), anti-Tra1-81 (Millipore), anti-TroponinT (Thermo Scientific, MA, USA), anti-alpha-actinin (Sigma-Aldrich), anti-GATA4 (Santa Cruz) and anti-ANP (Santa Cruz). Preparations were incubated with secondary antibodies for 1 h at room temperature. Nuclei were counterstained with 50 ng/ml 4′,6′-diamidino-2-phenylindole (DAPI; Invitrogen). Fluorescent signals were detected using a fluorescence laser microscope equipped with 1.5×10⁵ pixels charged coupled device (CCD) camera (BZ-9000, Keyence, Osaka, Japan).