Nkx2.5 antibody

Immunofluorescence assays were performed as follows: cells (1 × 10⁵ cells/well) were seeded into 12-well plates with a round coverslip (Biosharp) and cultured for 12–24 h. The cells were then fixed with 4% paraformaldehyde (YuanYe Bio-Technology) for 20 min at room temperature and then permeabilized with 0.5% Triton X-100 (Solarbio) for 15 min at room temperature. Nonspecific antibody binding was blocked by washing the cells with 5% normal goat working serum (Solarbio) for 1 h at room temperature, and the cells were incubated with NKX2.5 antibody (1:200, Abcam), GATA4 antibody (1:100, Proteintech), β-catenin (1:500, Proteintech), and cTnT antibody (1:500, Abcam) overnight at 4 ℃. The secondary antibody (Alexa Fluor 568, Invitrogen) was incubated with the cells at room temperature for 1.5–2 h, and DAPI (1:5000, Beyotime Biotechnology) was added for 10 min to stain the nucleus. Images were acquired using a fluorescence microscope (Carl Zeiss).

Immunofluorescent staining was performed on paraffin vertical sections using p-histone H3 (PH3) and Nkx2.5 antibodies. Briefly, the vertical sections were de-waxed in xylene, rehydrated and then heated in a microwave for antigen retrieval before exposure to the primary antibody with citrate buffer (pH = 6.0). Unspecific immunoreactions were blocked using 5% inactivated goat serum in PBS for 30 min at room temperature. The sections were washed in PBS and incubated with rabbit polyclonal PH3 antibody (1:200, Santa, Santa Cruz, CA, USA) or rabbit polyclonal Nkx2.5 antibody (1:200, Abcam, New Territories, HK) overnight at 4°C. Following extensive washing, the sections were incubated in goat anti-rabbit IgG secondary antibody that was conjugated to Alexa Fluor 555 dyes (1:500, Invitrogen, Waltham, MA, USA) for three hours at room temperature in a dark box. After immunostaining, all of the sections were counterstained with DAPI (1:1000, Invitrogen, Waltham, MA, USA) for 30 min at room temperature.