4.9. Histological Stainings

Different histological stainings were performed to determine the impact of eif4ebp3l overexpression during heart regeneration. The Acid Fuchsin Orange G (AFOG) staining was done as described [6]. The ventricle and infarct area were measured using Zen 3.1 software (blue edition, Carl Zeiss microscopy GmbH, Oberkochen, Germany). The percentage of infarct size relative to the ventricle as well as the percentage of fibrin and collagen were calculated.

For immunofluorescence, cryosections were immersed in boiling citric acid buffer (10 mM) for 30 min at 96 °C–99 °C for antigen retrieval. After short washes in PBT, the sections were blocked in 5% BSA, 5% NGS, 5% donkey serum, 0.5% Triton X-100, 20 mM MgCl₂ in PBS (Blocking buffer) for 1 h at room temperature. The sections were incubated with the antibodies anti-PCNA PC10 (mouse; abcam, 1:10,000) and anti-Mef2 (rabbit; boster biological technology, 1:200) in sequence overnight at 4 °C. Following washes with PBS, the sections were incubated with the secondary antibodies Alexa 647 (goat; invitrogen, 1:200) and Alexa 555 (donkey; invitrogen, 1:200) for 2 h at room temperature. Finally, the slides were mounted with DAPI mounting medium (Carl Roth, Karlsruhe, Germany). For quantification Mef2+ and Mef2+/PCNA+ cells were counted manually in a range of 150 µm from the border area of the infarct. To determine the percentage of proliferating CMs (considered as Mef2+/PCNA+ cells), five regions of interest (ROIs) were averaged in the border area of each heart.