Intravitreal Injection and Retinal Ganglion Cell Quantification in Mice

9- to 12-week-old male and female C57BL/6J (Charles River, UK, RRID:IMSR_JAX:000664), or Sarm1^-/- (RRID:MGI:3765957) mice were intravitreally injected with either 2 μL vacor, DMSO (vehicle) or PBS. Injections were performed as previously described (Osborne et al., 2018 (link)). ERG responses were recorded simultaneously from both eyes using an Espion E3 system with full-field Ganzfeld sphere (Diagnosys, Cambridge, UK). Animals were dark-adapted overnight, and ERG recordings performed under low level, red light illumination. Peak RGC responses (pSTR) were recorded between 70–110 ms after a light exposure of –4.73 log cd.s.m^–2, B wave peaks between 80–120 ms, at –1.90 log cd.s.m^–2, and A wave troughs within the first 20 ms at 1.29 log cd.s.m^–2. Following culling via exposure to CO₂, eyes were post-fixed for 24 hr in 4 % PFA before retinal flatmounts were prepared as previously described (Osborne et al., 2018 (link)). Briefly, retinas were excised from the eye cup, flattened and stained with the RGC specific nuclei marker BRN3A (Santa Cruz, sc-31984, RRID:AB_2167511, 1:200). Images were captured, blindly, from eight regions per retina using a 20 X objective, and automatically quantified with the Fiji plugin ‘Simple RGC’ (Cross et al., 2021 (link)). The representative images shown were acquired on a confocal microscope (Leica Microsystems) using a 40 X objective.