Immunostaining and Calcium Imaging for Cardiac Cell Characterization

Alkaline phosphatase (AP) was revealed using AS-BI phosphate-based detection (Millipore, SCR004). For LacZ visualization, samples were fixed and X-gal-stained overnight. Immunostaining was performed using anti-α-actinin (Sigma A7811) 1:200, anti-connexin 43 (Millipore AB1728) 1:50, anti-myosin heavy chain (MHC, Abcam) 1:250, anti-myosin light chain 2a (MLC2a, Synaptic Systems 311011) 1:250, anti-cardiac troponin T (cTnnT, Thermo 1:200) and Alexa 488 conjugated anti-myosin heavy chain (MF20, eBiociences 1:100) primary antibodies. Secondary antibodies (Invitrogen), goat anti-mouse IgG Alexa Fluor 568 (A11031) and goat anti-rabbit IgG Alexa Fluor 488 (A11008), were used at 1:250 dilution. Nuclei were labeled with 4,6-diamidino-2-phenylindole (DAPI; Invitrogen). Images were acquired by laser confocal microscopy (Zeiss LSM 510 Axiovert). Calcium dynamics were tracked on a Zeiss LSM live 5 confocal microscope after cells were loaded with Fluo-4 AM (Invitrogen) for 15 min at 37°C.^{28 (link)}