Staining of heart sections was performed as previously described (54). Briefly, embryos or adult hearts were fixed overnight at 4°C with 4% paraformaldehyde, washed with PBS. The samples were dehydrated using ethanol 50%, 70%, and 100% and then xylene prior to paraffin embedding. The embryos were cut with a microtome. After rehydration, heart sections were stained with H&E or Sirius red, or permeabilized with PBS 0.1% Triton X-100 and blocked with PBS 0.1% Tween 20 with 10% donkey serum; mouse anti-sarcomeric α−actinin antibody (1/500, Sigma-Aldrich, catalog H7811), was added in PBS 10% donkey serum and 0.1% Tween 20. The secondary antibody Alexa Fluor 488 was added in PBS 0.1% Tween 20.

Histopathological analysis of embryonic hearts was performed using ImageJ software (NIH).

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