Retinal Immunostaining and Analysis

Retinae and primary RGCs immunofluorescence analysis was performed as previously described [6 (link), 33 (link)]. In brief, retinal sections or cells were incubated with primary antibodies (1:100 diluted in 1% Normal donkey serum in PBS) at 4 °C overnight, followed by incubating with secondary antibodies (1:100 in PBS) at 37 °C for 1 h. Nuclei were counterstained with DAPI. Images were obtained by a Carl Zeiss LSM880 Meta confocal microscope. Statistical quantitative analyses of retinal immunostaining were performed as previously described [33 (link)]. Briefly, the intensity of immunofluorescence staining was quantified by measuring the area of ganglion cell layer (GCL) and inner plexiform layer (IPL) under the curvilineal diagrams plotted with Origin 8.0 software. Data from three independent curvilineal diagrams were averaged for each eye.