Mouse anti ctnt

After post-ablation imaging, cardiac disks were fixed in 4% PFA for 24 h. Samples for cryosection were treated with 30% sucrose for 24 h prior to OCT embedding and then sectioned at a thickness of 5-7 µm. Before staining, samples were incubated with 0.2% Triton-X-100 for an hour followed by another 2 h incubation with the blocking buffer containing 5% BSA, 0.1% Triton X-100, 2% goat serum, and 1% glycine. Cardiac troponin T (cTnT) was stained by the primary antibody mouse anti-cTnT (1:200 dilution, cat#MS-295-P1, ThermoFisher, Waltham, MA, USA) and the corresponding secondary antibody goat anti-mouse AlexaFluor647 (1:500 dilution, cat#A21236, ThermoFisher, Waltham, MA, USA). Cell nuclei were stained with DAPI. Sectioned samples were also stained for connexin 43 (CX43) using rabbit anti-Cx43 (1:100 dilution, cat#ab11370, Abcam, Cambridge, UK) and goat anti-rabbit AlexaFluor488 (1:500 dilution, cat#A11008, ThermoFisher, Waltham, MA, USA). Samples were examined using a DMi8 fluorescence microscope (Leica, Wetzlar, Germany). To determine the fiber length of cTnT proteins, five 20× images were obtained from each condition and analyzed by using the Tubeness plugin in Fiji (ImageJ; National Institutes of Health, Bethesda, MD, USA) to identify fiber structures first and then using the Anamorf plugin [60 (link)] in Fiji to measure fiber length.

Reprogrammed iCMs were harvested at DPI-7 and fixed with 4% PFA. For immunostaining of αMHC-GFP and cardiac troponin-T (cTnT), cells were incubated with conjugated antibodies of rabbit anti-GFP-FITC (1:400, Invitrogen, Carlsbad, CA, USA) and mouse anti-cTnT (1:400, Thermo Scientific, Waltham, MA, USA) at 4 °C overnight, and then incubated with fluorescence conjugated secondary antibodies (Invitrogen). Cells were analyzed by BD Accuri C6.