Sc 8697

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).

Mouse embryos or embryonic hearts were harvested at different stages and fixed in ice-cold PBS containing 4% paraformaldehyde overnight at 4°C. Fixed embryos were washed with PBS, then saturated in a 5, 10, 15, and 20% sucrose series in PBS, embedded in OCT Tissue-Tek mixed with 20% sucrose (1:1), and cut into 8-μm sections using a Leica CM 3050S cryostat (Leica Microsystems). Sections were permeabilized with wash buffer (PBS with 0.2% Triton 100), then blocked with blocking buffer (PBS with 1% BSA, 5% donkey serum, 0.2%Triton 100) for 1h at room temperature, followed by overnight incubation at 4°C with primary antibody diluted in blocking buffer. Sections were rinsed in wash buffer and incubated with fluorescently conjugated secondary antibodies and DAPI diluted in blocking buffer at room temperature for 1 hour, before rinsing and mounting in mounting buffer (Dako). Sections were imaged using an Olympus Fluoview FV1000 confocal microscope. Primary antibodies included: OGT (1:200, 61355, Active Motif), RL2 (1:300, MA1-072, Thermo Fisher), CD31 (1:150, 550274, BD Biosciences), NKX2-5 (1:50, SC8697, Santa Cruz Biotech), smooth muscle myosin heavy chain (1:200, BT-562, Alfa Aesar) and alpha actinin (1:400; 650931, Sigma), HIF1α (NB100-479, 1:200), TroponinT (Thermo Scientific, clone 13–11, 1:100).