Pcs sp5

Cultured cells were fixed with 4% paraformaldehyde in PBS, blocked in 0.3–0.6% Triton X‐100 with 1% bovine serum albumin for 40–60 minutes, and then incubated with primary antibodies overnight at 4°C. Primary antibody information is shown in Appendix Table S2. Secondary antibodies tagged with Cy3 (1:200, Jackson Immunoresearch Laboratories, West Grove, PA, USA) or Alexa Fluor 488 (1:200, Life Technologies) were used for visualization. The stained cells were mounted using VECTASHIELD with DAPI mounting solution (Vector Laboratories, Burlingame, CA), and images were obtained with an epifluorescence microscope (Leica, Heidelberg, Germany) and confocal microscope (Leica PCS SP5).
To analyze cell maturation, total fiber length, soma size, and intensity, the cells were measured using an image analysis system (Leica LAS).

Cells were seeded for 16–24 h into sterile 8-wells Chamber Slides (Falcon). After treatments, cells were fixed with 2% paraformaldehyde for 10 min, permeabilized in iced methanol and incubated with 3% BSA for 30 min (Sigma #A2153). After incubating with Ab against cardiac myosin, troponin T, c-Myc, KLF-4 and Nanog (see Table 1) at 4°C for 18 h, cells were washed with PBS followed by incubation with Alexa 488 anti-rabbit secondary antibody (1:500, Molecular Probes) and DAPI 1:1000 for 20 min. Coverslips were mounted in ProLong® Gold Antifade reagent (Life technologies #P36930) and examined using a confocal microscope Leica PCS SP5.