Evos fl digital inverted fluorescent microscope

Single cardiomyocytes or hiPSCs were cultured on matrigel-coated tissue culture plates (Falcon), and subsequently fixed in 4% PFA for 15 minutes at room temperature. Cells were blocked and permeabilized in 2% BSA, 2% FBS, and 0.01% Triton for 1 h at room temperature. Primary antibodies anti-human cTNT (1∶100, ThermoScientific), MLC2v (1∶200, Synaptic Systems), OCT4 (1∶100, BioVision), and SSEA4 (1∶25, Developmental Studies Hybridoma Bank) were added to blocking/permeabilization buffer overnight at 4°C. Secondary antibodies were either goat-anti-mouse or anti-rabbit AlexaFluor 488 or 594 (1∶400, Invitrogen). For TRA1-81 live cell immunostaining, hiPSCs were incubated with anti-human TRA1-81-Biotin (1∶100, eBioscience) for 2 h at 37°C in hESC media. Primary antibody was detected by incubation with PE-conjugated streptavadin secondary antibody (1∶100, eBioscience) for 2 h at 37°C in hESC media. Fluorescence was detected on the EVOS FL digital inverted fluorescent microscope (Life Technologies).