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Sequestration of extracellular Prox1 by anti-Prox1 antibody restores MG proliferative potential. a Schematic representation of Prox1 depletion in MG through sequestration of extracellular Prox1 protein by αProx1. b C57BL/6 J mice received intravitreal injections of AAV2-Ctrl Ab or AAV2-αProx1 at a concentration of 5 × 109 genome copies/eye. Mice were subsequently injected with MNU to induce retinal injury and EdU to label newly generated cells following MNU injury. c Retinas of the infected mice were subjected to immunostaining using mouse anti-FLAG antibody to detect FLAG-tagged proteins. Additionally, the distribution of Prox1 in the retinas was assessed by co-immunostaining with rabbit anti-Prox1 antibody. The MG identity of Prox1-positive cells was also determined through co-immunostaining with goat anti-Sox2 antibody. Enlarged views of boxed areas from the top row are presented in the second row and further magnified in the bottom rows, with Sox2-positive MG nuclei outlined by dotted lines. d Relative Prox1 immunofluorescent intensity in MG in the corresponding retina, normalized to Prox1 intensity in BCs within the same image, is shown. Each dot represents the median intensity collected from one retina. Numbers of samples analyzed are shown in the graph (data from 4 independent litters). e The identities of EdU-labeled cells in the retinas were determined by co-staining of Sox2 and Iba1. The boxed areas in the top row are magnified in subsequent rows. Arrowheads indicate EdU-labeled cell nuclei. (f) Quantification of EdU-labeled MG and microglia in the retinas is shown in the graph. Numbers of samples analyzed are shown in the graph (data from 4 independent litters). g Composition of EdU-labeled cells in the mouse retinas is displayed in the graph. Error bars denote SEM. P-values were calculated using one-sided Student’s t-test (*, p < 0.05; ***, p < 0.005; ****, p < 0.001; n.s., >0.05).
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