Under anesthesia, a 2-cm incision was created in the animal’s scalp and two small holes were drilled into the skull as illustrated [8 (link)]. Next, injections of 10-μl of 5% fluorogold (Sigma-Aldrich) were carried out using a micropipette at 3.8, 4.0, and 4.2 mm below the surface. The fluorogold was injected 3 days before the animals were sacrificed. Retrieval, fixation, dissection and processing of retinal samples were performed as described previously [8 (link)]. The RGC density was calculated as the ratio of the total number of RGCs divided by the total area of the retinal sample [8 (link)].
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