Axio imager z1 apotome microscope with mrm digital camera

The thickness of the porcine corneal epithelium was measured with an image analyzing system (ZEN Imaging Software, Carl Zeiss, Oberkochen, Germany). To reduce test variability, the mean of six measures per picture and six pictures of 200-fold magnification per sample was used for analysis of porcine corneas. The Zeiss Axio Imager Z1 Apotome Microscope with Mrm digital camera (Carl Zeiss, Oberkochen, Germany) was used for imaging.

Corneal epithelial cell death was analyzed using fluorescent in situ terminal deoxynucleotidyl transferase (TdT)-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL assay, Roche Diagnostics, Mannheim, Germany). TUNEL assay was performed according to the manufacturer’s protocol, as described previously [57 (link)]. To stain the nuclei, 4′,6-diamidino-2-phenylindole (DAPI) staining was performed. The Zeiss Axio Imager Z1 Apotome Microscope with Mrm digital camera (Carl Zeiss, Oberkochen, Germany) was used for imaging. A cell count was performed with semi-automated counter software ImageJ (ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA, https://imagej.nih.gov/ij/, 30 November 2021). To reduce test variability, the mean of six picture per sample of 200-fold magnification was used for analysis (n = 8 for 12 h LH/n = 8 for 12 h HH/n = 4 for 24 h LH/n = 6 for 24 h HH/n = 9 for 48 h LH/n = 6 for 48 h HH).