Retinal Whole-Mount Iba-1 Immunostaining

In order to label microglia (and potential macrophages infiltration from the circulation), an Iba-1 immunostaining was performed on retinal whole-mounts^{30 (link)}. Mice were deeply anaesthetized (i.p. 30 mg/kg sodium pentobarbital, Nembutal) and sacrificed by cervical dislocation. Eyes were dissected and fixed for 1 hour in 4% PFA. Next, retinas were whole-mounted and again fixed for 1 hour in 4% PFA. Whole-mounted retinas were frozen for 15 minutes at −80 °C and rabbit anti-Iba-1 (Wako Chemicals, #019-19741) (1:1000), diluted in 10 mM phosphate-buffered saline (PBS) containing 2% Triton X-100 and 2% goat pre-immune serum, was applied overnight. The next day, a secondary goat anti-rabbit IgG antibody conjugated to an Alexa fluorophore-488 (Life Technologies) (1:200) was applied for 2 hours. Retinal whole-mounts were rinsed with PBS with 0.5% Triton X-100 in between steps, and mounted using mowiol mounting medium (10% mowiol 4–88, 40% glycerol, 0.1% 1,4-diazabicyclo-[2,2,2]-octane in 0.2 M Tris-HCl [pH 8.5]). Mosaic z-stack images (step size 3 μm; 20–30 stacks per image, comprising the retinal nerve fibre layer till the photoreceptor layer; 20x objective; 1 pixel = 1.24 μm) of the entire whole-mount were taken with a laser confocal scanning microscope (FV1000, Olympus), controlled with FluoViewer 4.2 software (Olympus), and a maximum intensity projection was made for further analysis.