Immunostaining Protocol for Retinal Ganglion Cells

Immunostainings for Brn3a, GFP and RBPMS on retinal whole-mounts were completed as described previously^{22 (link),28 (link),29 (link)}. The primary antibodies used are: mouse anti-Brn3a for single labelling (1:500, Merck Millipore, Darmstadt, Germany); goat anti-Brn3a for co-labelling with GFP or RBPMS (1:750, Santa Cruz, Dallas, TX, USA); rabbit anti-GFP (1:10,000, in-house made); and rabbit anti-RBPMS (1:200, PhosphoSolution, Aurora, CO, USA). Immunolabeled RGCs were examined under confocal microscopy (LSM 710, Carl Zeiss MicroImaging GmbH, Jena, Germany). Each retinal whole mount was imaged as a tiled z-stack at × 10 magnification, which was used to generate a single plane maximum projection of the RGC layer in each retina for subsequent analysis. Retinal image acquisition settings were kept constant for all retinas imaged, allowing comparison of Brn3a expression in each experimental group as previously described³⁰ . Each whole mount image was manually orientated, using in vivo cSLO imaging of retinal vasculature as a reference, so that the superior retina was towards the top of the image.