Wild-type adult male mice were anesthetized and placed in a stereotactic apparatus (Stoelting, Kiel, WI, United States) as we did before (Lin et al., 2012 (link)). The skull was exposed and cleaned with 3% hydrogen peroxide. The accurate location of superior colliculus was conducted followed mice brain atlas (The Mouse Brain in Stereotaxic Coordinates, Academic Press, New York, 2001). A hole of 1 mm in diameter was drilled in the skull (4.4 mm posterior and 0.06 mm lateral to the bregma), and a 26-gauge stainless steel cannula was inserted for infusion of fluorochrome, hydroxystilbamidine (FG; Biotium, Inc., Hayward, CA, United States) at a speed of 1 μl/10 min. One week before ON lesion. One microliter of 2% FG was injected into the bilateral superior colliculus (1.2 mm deep from the skull). To analyze the survival number of RGCs in whole retinas labeled with FG at 7-d post-ON crush (ONC7d), the golden dots (survived RGCs) were counted using FIJI software (NIH, United States).
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