2.5. Immunofluorescence Staining

For immunofluorescence staining, cardiomyocytes were cultured on fibronectin/gelatin-coated glass bottom dishes (MatTek Corporation, Ashland, USA). Cultured cells were washed with PBS and fixed for 6 min with diethyl ether/ethanol mixture (1 : 1) at -20°C. Fixed cardiomyocytes were washed with PBS and incubated with 1% FBS in PBS for 30 min. Primary antibodies were diluted in PBS, and cells were incubated for 2 h in a humidified chamber at room temperature. Goat anti-α-actinin (ACTN1) antibody (Novus Biologicals, Centennial, USA; #AF8279) and mouse anti-p21/CDKN1A antibody (Thermo Fisher Scientific; #AHZ0422) were used as primary antibodies. Afterwards, cardiomyocytes were washed with PBS and incubated with secondary antibodies conjugated to Alexa Fluor® 546 nm (Thermo Fisher Scientific; #A-11056) and 647 nm (Abcam, Cambridge, UK; #ab150107) for 30 min in a lightproof, humidified chamber at room temperature. Samples were covered with Roti®-Mount FluorCare including DAPI (Carl Roth, Karlsruhe, Germany; #HP20.1) as mounting medium.

Microscopic visualization was carried out using a confocal laser scanning microscope at 630-fold magnification (objective Plan-Apochromat 63x/1.40 Oil DIC M27). Cardiomyocytes were defined by their characteristic sarcomeric striations elucidated by ACTN1 staining. The cell area was calculated via Zen 2012 SP5 (black), LSM 780 (Carl Zeiss, Jena, Germany; v. 14.0.0.0). To determine p21 in neonatal cardiomyocytes, an overlap between DAPI and p21 stained nuclei in ACTN1-positive cells was analyzed using the software FIJI (v. 1.52n).