Evaluating Retinal Degeneration through Confocal Microscopy

Images of immunolabeled cryosections were acquired by using a Leica TCS SP5 (Wetlzar, Germany) or a Nikon 80i confocal microscope. The central dorsal retina was selected for the analysis, since retinal degeneration develops in that specific region called a hotspot [39 (link)]. The same parameters were set up for all the acquisitions. For the final images, ~22 planes at a distance of 0.5 µm were acquired. The fluorescence intensity of all markers was quantified through ImageJ software. Differences in immunofluorescence signals throughout the retinal layers (OS, ONL, OPL, INL, IPL, GCL) were investigated through ImageJ software and by using profile plots (range 0–50) with the corresponding grayscale intensities. For the LD + 7rec group, the outer retina was indicated as SUB/ONL (subretina/outer nuclear layer) since the degenerated tissue loses the physiological retinal architecture with the occurrence of rosettes and neovascularization from the choroid [26 (link)].