α tropomyosin

Antibodies for α‐Actin, troponin T, α‐tropomyosin, β‐tropomyosin, and laminin proteins were purchased from Sigma‐Aldrich Inc. Antibodies for troponin I and troponin C were purchased from Thermo Scientific Inc. Antibodies for β‐TUBULIN; myosin heavy (MHC) and light chain (MLC) isoforms were obtained from the University of Iowa Developmental Studies Hybridoma Bank.

All animal experiments were performed according to protocols approved by the animal welfare committee of the Leiden University Medical Center and conform the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. Female Rosa26^mTmG/mTmG mice were set-up for timed matings with male Wt1^creERT2/+. The presence of a plug in the morning was denoted as embryonic day (E)0.5. At E9.5, the mother was injected with tamoxifen (2 mg) to label the pro-epicardial cells. After three days, when the epicardium has covered the heart, embryos were isolated from which the embryonic heart was dissected and cultured based on a previously published protocol (Jackson-Weaver et al., 2020 (link)). Embryonic tissue was cultured in DMEM high glucose and M199 mixed in a 1:1 ratio supplemented with 0.1% FBS and 100 U/mL penicillin and 100 mg/ml streptomycin at 37°C in 5% CO₂. After 24 h, the culture medium of the embryos carrying the Cre + genotype was supplemented with 2 ng/ml FGF with or without 200 ng/ml Activin A and incubated for another 48 h. The tissue was fixed in 4% PFA, embedded in paraffin, sectioned, and stained as described (Kruithof et al., 2020 (link)) using the following antibodies: α-GFP(Abcam, ab13970), α-Tropomyosin (Sigma-Aldrich, T9283) and α-Wt1 (Abcam, ab89901).