Ab56357

Rat neonatal CMs were fixed in 4% paraformaldehyde. Anti-cardiac troponin I (CTNI) 1:500 (ab56357, Abcam) and anti-PKM2 1:100 (D78A4, Cell Signaling) were used. After washing with PBS, samples were stained with Alexa-568, Alexa-488 or Alexa-647 secondary antibodies 1:500 (Life Technologies), followed by 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) 1:2000 (Merck) staining to visualize DNA.

For immunostaining, E8.5 mouse embryos were fixed with 4% paraformaldehyde. Antibodies against ASB2 1:100 (HPA001546, Sigma) and anti-cTnI 1:500 (ab56357, Abcam) were used. For western blotting, E8.5, E14.5, P1 and P7 mouse hearts were isolated. Antibodies against TCF3 1:1,000 (G-2, Santa Cruz Biotechnology) were used.

One LV short-axis section (10 μm thickness) at the level of the mid-lower papillary muscles was chosen for each heart. First, the slides were washed with PBS and fixed with 4% PFA for 10 min. The samples were then permeabilized with 0.5% PBS-Tween for 15 min, followed by blocking for 1 h with 10% fetal bovine serum. The sections were then stained with anti-cardiac troponin I primary antibody (1:200, Abcam, Cat: ab56357), along with a primary antibody for Ki-67 (1:100, Abcam, Cat: ab15580) or aurora B kinase (AurB, 1:100, Abcam, Cat: ab2254) for 90 min at 37 °C. The slides were then washed in PBS, and appropriate secondary antibodies (1:200, Abcam, Cat: ab150064 or ab150129) were applied for 45 min. Finally, the sections were incubated with DAPI (NucBlue Fixed Cell ReadyProbes Reagent, ThermoFisher Scientific, Cat: R37606) for 5 min and washed again. Microscope coverslips (Fisherbrand, Cat: 12545F, Pittsburgh, PA, USA) were added and the slides were kept at 4 °C until ready for imaging.

Tissues were fixed in 4% paraformaldehyde overnight, cryoprotected by overnight incubation in 30% sucrose, and embedded in tissue freezing medium (VWR, 25608-930). 8 µm cryosections were obtained using a cryostat (Thermo Scientific, Micron HM 550). Sections and cells were incubated in blocking medium (PBS containing 5% donkey serum, 0.2% Triton X-100) at 4 °C overnight. For immunostaining, sections were incubated with primary antibodies at 4 °C overnight and secondary antibodies for 2 h. Primary antibodies used were as follow: aYAP1 (1:200, Abcam, ab205270), Ki67 (1:200, Abcam, ab16667), γ-H2AX (1:200, Abcam, ab81299), ACTN2 (1:200, Abcam, ab137346), cardiac troponin I (1:200, Abcam, ab56357), ATP5B (ATP synthase, H⁺ transporting mitochondrial F1 complex, beta subunit; 1:200, Abcam, ab14730), MFN1 (1:200, Abcam, ab104274), DRP1 (1:200, ProteinTech, 12957-1-AP), and 4HNE (1:200, Abcam, ab46545). After washing with blocking buffer, the slices were incubated with optimal secondary antibodies with/without WGA and/or DAPI at room temperature for 2h. After washing, samples were mounted with ProLong Diamond antifade mountant (Invitrogen, 36961) and imaged using an Olympus FV1000 confocal microscope. Laser scanning confocal microscope (Olympus, FV1000) were taken to determine confocal fluorescence images. Fluorescence intensity and cell size were measured by ImageJ.