Ab45932

Cardiomyocytes were seeded on glass coverslips and fixed with 4%
paraformaldehyde. Cells were permeabilized with 0.1% Triton-X (Sigma T8787), and
a blocking step was performed with 4% swine serum (Jackson Immunoresearch,
#014-000-121) for 1 h at room temperature. Primary and secondary antibodies were
diluted in 4% swine serum and incubated at room temperature for 1 h. Nuclei were
stained with DAPI. Imaging was performed with the LEICA TPS SP8X. Image
visualization and processing was performed with LAS-X (Leica) software. The
following primary antibodies and dilutions were used TNNT2, 1:1000 (Abcam,
#ab45932); ACTN2, 1:800 (Sigma Aldrich, #A7811), SHOX2, 1:200 (Abcam, #ab55740),
NR2F2, 1:200 (R&D Systems, #PP-H7147-00), MYL2, 1:200 (Abcam, #ab79935).

Immunocytochemical staining were performed on cells seeded onto Geltrex coated optical tissue culture chamber (Thermo Scientific Lab-Tek II Chamber Slide System, 154,526). Cells were fixed in 4% paraformaldehyde (PFA) in PBS for 15 min at RT followed by PBS washings. For staining, fixed cells were permeabilized with 0.2% Triton X-100 (Fisher Bioreagents, BP151) in PBS for 10 min prior to blocking non-specific binding with 10% normal serum in PBST (PBS with 0.1% Tween-20 (MP Biomedicals, 11TWEEN201) for 30 min. Cells were incubated with primary antibodies at 4°C overnight and then stained with secondary antibodies at room temperature for 1 h and mounted with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, H-1200). The stained cells were imaged with a Leica TCS SP5 microscope using LAS X software (Leica Biosystems) or an LSM 880 with Airyscan Confocal Microscope using ZEN software (Carl Zeiss Microscopy). The following primary antibodies were used: Rabbit anti-Cardiac Troponin T (Abcam, ab45932,1:400), Mouse anti-cardiac ACTN2 (Sigma, A7811, 1:300), Rabbit anti-TOMM20 (Abcam, ab2043078, 1:1,000), Mouse anti-dsDNA (Abcam, ab470907, 1:1,000).