Retinal Immunocytochemistry and Imaging

Mice were sacrificed, eyes enucleated and fixed overnight in 4% paraformaldehyde in PBS before the retinas were removed from the eyecups and immunocytochemistry performed as described previously described [58 (link)]. Whole retinas were incubated with anti-BRN3A primary antibody (Synaptic Systems 411003, Goettingen, Germany; 1/200 dilution) [59 (link),60 (link)] for 3 days at 4 °C, washed in PBS and then incubated with Cy3 conjugated secondary antibodies (Jackson ImmunoResearch Laboratories; 1/400) for 2 days at 4 °C. Wholemount and cell images were taken using an Olympus IX83 inverted motorised microscope (Mason Technology, Dublin, Ireland) equipped with a SpectraX LED light source (Lumencor, Mason Technology, Dublin, Ireland) and an Orca-Flash4.0 LT PLUS/sCMOS camera (Hamamatsu, Tsukuba City, Japan), as previously described [47 (link)]. Samples were imaged using a 4x (wholemount) and 40× (cells) objective utilising enhanced focal imaging (EFI) with typically 5–8 Z-slices. Lateral frames for wholemounts were stitched together and analysed in Olympus CellSens software (v1.9; Waltham, MA, USA). Cell counting was performed utilizing 2D deconvolution, manual threshold and object size filter, with the same settings/operations applied to all images.