Evos xl light microscope

There were n = 3 Sprague-Dawley rats for each group (i.e., control, MEA implantation, and sham). Control group did not receive any surgery, and hearts were collected directly. Hearts from the MEA implantation group were collected at 2, 4, 6, and 9 weeks after implantation surgery. Hearts from the sham group were collected at 2 weeks after the sham surgery. Rats were euthanized using 5% isoflurane vapors at oxygen flow of 2 ml/min with an EZ anesthesia machine (EZ Systems Inc.) until loss of consciousness was confirmed via toe pinch. Hearts were excised and retrogradely perfused via an aortic cannula with cardioplegic solution (110 mM NaCl, 16 mM KCl, 16 mM MgCl₂, 10 mM NaHCO₃, and 1.2 mM CaCl₂; 4°C) and then 10% neutral-buffered formalin. Hearts were transferred to a 70% ethanol solution after 24 hours of immersion (room temperature) in 10% neutral-buffered formalin. Cross sections of hearts were paraffin-embedded, sectioned, and stained with Masson’s trichrome for identification of myocardium (red color), fibrosis (blue color), and interstitial space (white color). Tissue samples were imaged using an EVOS XL light microscope (Thermo Fisher Scientific). A custom MATLAB code was used to quantify the percent volume of cardiomyocytes, fibrosis, and interstitial space in the images (i.e., calculate the relative number of pixels per color in the selected region of interest).