Alexa fluor 563

iPSCs were harvested using Versene 0.02% EDTA (Life Technologies) and were seeded on Matrigel coated coverslips and were let to grow for 5 days in mTeSR1. Cells were washed with 3 X PBS, fixed using 4% parafomaldehyde (15 mins), washed and then permabilized with 0.2% Triton-X (15 mins), washed and blocked for 3 hours in 3% goat serum and incubated with Tra 1–60 (Abcam) and NANOG (Abcam) overnight in 4 °C. Cells were washed with 3 X PBS and incubated for 1 hour with secondary antibodies Alexa-Fluor 488 (Abcam) and Alexa-Fluor 563 (Abcam). Cells were washed with 3 X PBS and mounted on a microscope slide with antifade medium containing DAPI (VECTASHIELD, VECTOR laboratories).

Cells were seeded on Matrigel-coated coverslips and were left to attach for 2 days. Cells were washed with 3 X PBS, fixed with 4% parafomaldehyde (15 mins), washed and permabilized with 0.2% Triton-X (15 mins), washed and blocked for 3 hours in 3% goat serum and incubated with cTnT (Abcam) and sarcomeric α-actinin (Abcam) overnight. Cells were washed with 3 X PBS and incubated for one hour with secondary antibody Alexa Fluor-563 (Abcam) and Alexa Fluor-488 (Abcam) and were then washed and mounted on a slide using Antifate mounting medium containing DAPI (VECTASHIELD, VECTOR laboratories). Slides were viewed on Zeiss LSM880 confocal microscope.