Mice were sacrificed 3 days after OKR assessment and eyes were enucleated and fixed in 4% paraformaldehyde in PBS overnight. Eyes were washed in PBS, then the retinas were removed from the eyecups and immediately processed for immunocytochemistry. Immunocytochemistry was performed as described previously (Palfi et al., 2016 (link)). Whole retinas were incubated with primary antibodies for RBPMS (ABN1376, Millipore, 1:200; Rodriguez et al., 2014 (link)) overnight for 3 days at 4°C. Retinas were then washed in PBS and incubated with secondary antibodies conjugated with Alexa-Fluor-488, Cy3 (Jackson ImmunoResearch Laboratories; 1:400) for 2 days and nuclei counterstained with DAPI. Samples were covered using Hydromount (National Diagnostics). Fluorescent microscopy was carried out utilizing an Olympus IX83 inverted motorized microscope (cellSens v1.9 software) equipped with a SpectraX LED light source (Lumencor) and an Orca-Flash4.0 LT PLUS/sCMOS camera (Hamamatsu). Samples were imaged using a 10x plan fluorite objective utilizing enhanced focal imaging (EFI) with typically 5–8 Z-slices. Lateral frames were stitched together and analyzed in cellSens. Automated cell staining area was calculated utilizing 2D deconvolution, manual threshold and object size filter in cellSense; the same settings/operations were applied to all images.
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