*2.5. Immunofluorescence Imaging and Quantification*

Imaging of each full section was done with the Opera Phenix ™ high-content screening system and Harmony software (Perkin Elmer, Montreal, QC, Canada) with a field overlap of 5%, at a 40× magnification. The images were further analyzed using Columbus ™ system (Perkin Elmer, Montreal, QC, Canada) to quantify the signal of the immunofluorescent staining. Analyses were applied which recognized DAPI as a nucleus marker and SRC as a cytoplasmic marker to segmen<sup>t</sup> the cells for quantification of fluorescence intensity in each cellular compartment. The 4-HNE and 8-OHG intensities were used to determine oxidative damage in membranes and DNA, respectively. Analyses were done separately in the caput, corpus and cauda epididymides, as well as in the epithelium and in the interstitial cells (endothelial, smooth muscle, and conjunctive cells).
