Quantification of microglial cells and RPE morphology in aged DKO RPE/choroid flatmounts

Five fluorescent images (40X objective lens) were taken from each RPE/choroidal flatmount. The number of microglial cells and the morphology of RPE in aged DKO mice were quantified. 6 eyes per group were used in this study. FIJI ImageJ plug-in Bio-voxxel software [40] was used to outline the shape of each RPE cell. For each RPE cell, the area and perimeter were measured using FIJI and the shape factor was calculated by the eq. 4πArea/Perimeter2 [41]. The shape factor value ranges from 0 to 1, with one indicating a perfect circular cell, and zero indicating an elongated cell. The shape factor of a perfect hexagonal cell (eg. a RPE cell) is around 0.9 [42]. The relationship between microglial cell number and the shape factor of the RPE was analyzed by linear regression using SPSS statistics software (IBM, Chicago, IL, USA). The quantification was confirmed by two independent researchers.

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