Ab32107

Freshly-dissected embryo bodies were observed under a stereomicroscope. Parts of the embryos and embryonic hearts were fixed overnight with 4% paraformaldehyde (PFA) at 4°C and then embedded in paraffin wax or optimal cutting temperature compound (OCT). The paraffin sections were stained with H&E for detection of the cardiac structure. To analyze cell proliferation and detect apoptosis in the cardiac OFT, sections from HO or WT individual embryos at E13.5 were stained with anti-PHH3 (ab32107, Abcam, Cambridge, UK) (45 (link)) and in situ Cell Death Detection Kit, Fluorescein (11684795910, Roche, Mannheim, Germany). For detection of the cNNCs, the OFT-containing sections were incubated with anti-Sox10 primary antibody (ab227680, Abcam, Cambridge, UK), washed with phosphate buffered saline solution with 0.1% Tween 20 (PBST), and then incubated with a secondary antibody [goat antirabbit IgG (H + L) (A27039, Invitrogen, Carlsbad, CA, USA)] in fluorescence immunohistochemical assay. Finally, slides were mounted using DAPI medium (SL1841, Coolaber, Beijing, China) and then three discontinuous sections per embryo subjected to fluorescence microscopy on a Leica microscope (DM6000B, Leica, Germany). Signals were quantified using (ImageJ software), with 10 fields that were randomly selected from three cardiac sections used to quantify positive cell number from the fluorogram.

AMTs were fixed using 4% paraformaldehyde/PBS for 30 minutes and embedded in 13% polyacrylamide gel. 150 μm thick sections were made using a vibratory microtome and stained for cardiac troponin-T (TNNT2, MS-295-P, Thermo Scientific, 1:150 dilution), alpha sarcomeric actinin (α-Actinin, EA53, Sigma, 1:200 dilution), connexin-43 (GJA1, ab11369, Abcam, 1:100 dilution), active caspase-3 (ab2302, Abcam, 1:100 dilution), NKX2-5 (sc-8697, Santa Cruz, 1:50 dilution), and phospho histone H3 (P-histone3, ab32107, Abcam, 1:100 dilution) primary antibodies and Alexa Fluor secondary antibodies. Stained samples were scanned using a confocal microscope and used to generate 3D projection images of the tissue sections in ImageJ. Proliferation and apoptosis were assessed by calculating the percentage of total nuclei positive for phospho histone H3 and active caspase-3, respectively. Myotubes were identified as actinin positive cells containing 5 or more nuclei, since human CMs can contain up to 4 nuclei70 (link).