Cardiac Tissue Imaging and Quantification

Image acquisition was performed using the following equipment: upright light microscope Leica DM500 (Leica Microsystems, Switzerland), epifluorescence microscope Olympus MVX10 (Olympus Corporation, Japan), and the confocal microscopes Zeiss LSM 510 and Zeiss LSM 710 (Carl Zeiss AG, Germany). Image analysis and quantification were performed using the software ImageJ^{77 (link)}. In the case of IHC-samples, at least two cryosections containing the most representative areas of the injured/regenerating tissue were considered for each heart ventricle. For the analysis, regions of interest were first digitally selected to cover the entire injured/regenerating tissue starting from the amputation plane. Then, in order to analyze the fluorescens signal, fixed parameters for particle size and shape (TUNEL⁺-nuclei, mpx⁺-cells) and pre-determined signal-thresholds (TUNEL⁺-nuclei, mpx⁺-cells, mpeg1⁺-cells, fli1a⁺-vessels) were selected to allow software-assisted quantification Cardiomyocyte proliferation was determined on blinded-samples, for which descriptions of the specific morphology of the cardiomyocytes and their nuclei^{79 (link)} were used as guidelines. For normalization, the results were divided by the area of the region of interest, which considered a margin of 300 µm above the amputation plane and the regenerating tissue beyond.