Tnnt2

Cell sheets differentiated for 34 days were dissociated into single cells with TryPLE and seeded at low density onto LN-521+LN-221coated plates. After 3 days, CMs were washed once with PBS and fixed with 4% paraformaldehyde (PFA) (Sigma, Cat.#28908) for 20 mins at 4 C. The cells were then washed with PBS and blocked with blocking buffer containing 1% BSA, 5% goat serum (Sigma, Cat.#G9023) and 0.1% Triton X (Sigma, Cat.#T8787) for 1 hr at room temperature. After blocking, cells were incubated with primary antibody: TNNI3 (Santa cruz, Cat.#sc-15368, 1:20), NKX2-5 (Santa Cruz, Cat.#sc-14033, 1:100), TNNT2 (1:200), a-actinin (Sigma, Cat.#A7811, 1:500) in PBS containing 1% BSA and 5% goat serum at room temperature for 1 hour. Following this, antibody solution was removed and cells washed thrice with PBS on a shaker for 5 mins each. Subsequently, the cells were incubated at room temperature with Alexa-conjugated secondary antibody (ThermoFisher, 1:1000) on a shaker at low speed in the dark for 1 hr. Finally, cells were washed with PBS twice (5 mins) and ProLongâ Gold Antifade Mountant with DAPI (ThermoFisher, Cat.#P36931) added to stain the nuclei. Cells were visualized immediately in Leica TCS sp8 confocal fluorescence microscope.