Goat anti nkx2

To assess the purity of the cells, we performed immunofluorescence studies. All the steps, unless otherwise indicated, were performed at room temperature. Cultured cells were fixed in 4% buffered paraformaldehyde (PFA) for 10 minutes and then washed in PBS three times. After a common permeabilization step in 0.2% Triton X-100 in PBS for 10 minutes and an additional three washes in PBS, one set of samples was treated as follows: nonspecific binding was blocked by incubation in 10% normal goat serum for 1 hour and then incubated overnight at 4°C in rabbit anti-Islet-1 (Abcam, Cat. # ab20670, 2 μg/ml) or rabbit anti-GATA-4 (Abcam, Cat. # ab61170, dil. 1 : 500). After three additional washes in PBS, samples were incubated for 1 hour in FITC-conjugated goat anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA, Cat. # FI-1000, 10 μg/ml). Another set of samples was blocked in 10% normal rabbit serum for 1 hour and then incubated in goat anti-Nkx2.5 (Santa Cruz, Cat. # sc-8697, dil. 1 : 50) overnight at 4°C. Then, samples were incubated in AlexaFluor® 488-conjugated rabbit anti-goat antibody (Invitrogen, Cat # A-11078, dil. 1 : 200) for 1 hour. All the samples were then washed three times in PBS and mounted with VECTASHIELD® antifade mounting medium with DAPI for nuclear staining (Vector Laboratories, Cat. # H-1200).

Samples were fixed using 4% PFA with 0.25% TritonX-100. The following primary antibodies were used: mouse anti-cardiac Troponin T (Thermo MS-295-P, 1∶200); mouse anti-myosin heavy chain (clone MF20, eBioscience 53-6503-82, 1∶200); mouse anti-sarcomeric α-actinin (clone EA53, Sigma A7811, 1∶200); rabbit anti-Isl1 (Abcam ab20670, 1∶100), rabbit anti-smooth muscle myosin heavy chain (Abcam ab53219, 1∶250), goat anti-Nkx2.5 (Santa Cruz Biotechnology sc-8687, 1∶100), chicken anti-vimentin (Millipore AB5733, 1∶500), mouse anti-Myl7 (Abcam ab68086, 1∶100), rabbit anti-Myl2 (Proteintech 10906-1-AP, 1∶100), rabbit anti-Ki-67 (Novus NB110-89719, 1∶100), and mouse anti-V5 (Invitrogen 46-0707, 1∶200). For Oil Red O staining, samples were incubated with isopropanol for 5 minutes, followed by a solution consisting of 3 parts Oil Red O solution (0.3% Oil Red O in 99% Isopropanol, Sigma) and 2 parts DI water for 5 minutes. After washing with water samples were stained with hematoxylin (Sigma), washed with water, and visualized.

Timed pregnant female mice received intraperitoneal injection of 100 mg/kg EdU one hour before sacrifice by isoflurane and cervical dislocation. E14.5 fetuses were isolated in 1xPBS on ice, the head was removed and the body fixed in 4% PFA for 24 hr. 7 μm sections were stained with mouse-anti-actin (1:400; Sigma A9357), goat-anti-nkx2.5 (1:150; Santa Cruz sc-14033), DAPI (1:1000) and EdU Click-IT (ThermoFisher, C10340) before imaging and counting. Nkx2.5 positive nuclei were designated cardiomyocytes, Nkx2.5 and EdU double positive nuclei were designated proliferating cardiomyocytes. Ratios were normalized within each litter.