Mice were anesthetized as described and exsanguinated by perfusion with oxygenated mammalian Ringer's solution containing lidocaine hydrochloride (0.1 mg/mL, Xylocaine; Astra USA, Inc., Westborough, MA) and heparin sodium (500 units/mL, Heparin; Elkins-Sinn, Inc., Cherry Hill, NJ). Transcardial perfusion was then continued with fixative (approximately 20 mL 4.0% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4). Retinas were dissected after identifying the inferior area of the retina by the inferior position of the ophthalmic artery. For immunohistochemical staining of retinal microglia, the retinas were incubated in a blocking solution containing 10% fetal bovine serum (FBS), and 0.5% Triton X-100 in PBS (pH 7.4). The primary antibody Iba1 was diluted 1:400 in 5% FBS, 0.4% Triton X-100. Retinas were incubated in primary antibody overnight in 4°C. Secondary antibody conjugated with Rhodamine red (1:400) was applied for 2 hours. The retinas were mounted in GB-mount (Golden Bridge International [GBI] Life Science, Inc., Mukilteo, WA). For immunohistochemical staining of RGCs, the retinas were incubated in a blocking solution containing 10% normal goat serum (NGS), 2% bovine serum albumin (BSA), and 0.5% Triton X-100 in PBS (pH 7.4). The primary antibody anti–β-tubulin TUJ1 was diluted 1:400 in 5% NGS, 1% BSA, 0.3% Triton X-100 in PBS and applied for 1 day in 4°C. Secondary antibody conjugated with Rhodamine red (1:400; Life Technologies, Grand Island, NY) was applied for 2 hours. The retinas were mounted in GB-mount (GBI Life Science, Inc.). The microglia and the RGCs were imaged at ×40 magnification by a confocal laser scanning microscope (Nikon C1 Confocal System, Tokyo, Japan).