*2.4. Immunofluorescent Labelling*

Retinas were transferred to slides and isolated using a hydrophobic barrier pen (VWR, Radnor, PA, USA). Subsequently, the tissue was permeabilized with 0.5% Triton X-100 (VWR) in 1 M PBS for 60 min and blocked in 2% bovine serum albumin (Thermo Fisher Scientific) in 1 M PBS for 60 min. Primary antibodies were applied and maintained overnight at 4 ◦C (detailed in Table 1). Retinas were washed five times for 5 min in 1 M PBS before the secondary antibodies were applied and maintained for 4 h at room temperature. All tissue was washed again five times for 5 min with PBS, and TOPRO-3 nuclear stain (1 μM in 1 M PBS) was applied and maintained at room temperature for 10 min. Tissue was then washed once in PBS and mounted using Fluoromount-G and glass coverslips (Thermo Fisher Scientific). Slides were sealed with nail varnish.


**Table 1.** Antibodies used.

#### *2.5. Analysis of Retinal Ganglion Cell Degeneration*

RGC loss and shrinkage of nuclei were evaluated by CFP, RBPMS, and TOPRO-3 labelling of flat mounted tissue. All images were acquired on a Leica DMi8 microscope with a CoolLED pE-300white LED-based light source and a Leica DFC7000 T fluorescence color camera (all Leica, Wetzlar, Germany). In each retina, six images (40× magnification, 0.55 NA) were taken at 0, 2, 4, 6, 8, and 10 o clock equidistantly, about a superior to inferior line through the optic disc (~1000 μm eccentricity). All images were cropped to 0.01 mm2. CFP+ cells, RBPMS+ cells, and TO-PRO-3+ nuclei were counted using the cell counter plugin for Fiji [10]. Cell counts and nuclei counts were averaged across the six images in each retina and expressed as a density per 0.01 mm2. To assess nuclear shrinkage, the nuclear diameter was measured in >30 nuclei belonging to RBPMS+ cells (per cropped image) using the in-built line tool, giving an average nuclear diameter per image. These values were averaged across the six images of each retina to produce a final average nuclear diameter per retina.
