Retinal Thickness Measurement Protocol

The detailed procedure has been published before^{20 (link),21 (link),25 (link)}. Briefly, after mice were perfused with ice cold 4% PFA in PBS, both eyes of each animal were enucleated and fixed in 1% PFA and 1.25% glutaraldehyde fixative prepared in 0.1 mM sodium cacodylate buffer with 5 mM calcium chloride and 5% sucrose for 24 h at room temperature. Globes were then dehydrated through a graded series of alcohols, infiltrated in propylene oxide, and embedded in epoxy. Sections 0.7micron in thickness were taken using an ultramicrotome (Reichert Ultracut E; Leica, Deerfield, Illinois) and stained with 0.5% toluidine blue. Slides were photographed by a light microscope (Eclipse E1000; Nikon, Tokyo, Japan). 20× images were used to measure the thickness of the inner and outer retina at the same distance from the optic nerve head. The mean of the inner or outer retinal thickness in the injured retina was compared to that in the contralateral control retina to yield a percentage of inner or outer retinal thickness value. The investigators who measured the thickness of inner or outer retina were masked to the treatment of the samples.