Western blotting was used to characterize the specificity of the RBPMS antibodies and to determine the presence of RBPMS in mouse and rat retina. Cell lysates (10 μg) from HEK293T cells transfected with human RBPMS cDNA was loaded alongside a lysate (10 μg) of non-transfected HEK293T cells (negative control) (OriGene Technologies) and assayed by a Western blot. Total protein (25 μg) from mouse and rat retina, and a rat retina collected at 56 days after optic nerve transection were also assayed by a Western blot. Prestained marker proteins were used as molecular mass standards (Bio-Rad, Hercules, CA). Proteins were fractionated (200V for 30 minutes) by 10% Tris-glycine SDS-Page using Mini-Protean TGX Gel System (Bio-Rad), rinsed briefly, and then transferred at 400 mA for 90 minutes at 4°C. Blots were blocked for 1 hour at room temperature in a non-mammalian Odyssey Blocker (Bio-Rad), and incubated with guinea pig or rabbit RBPMS antibodies (see Table 1) at 1:1500 overnight on a shaker at 4°C. Blots were rinsed in a solution containing 0.1 M PB, 0.154 M NaCl, and 0.05% Tween 20 (v/v) at pH 7.4 for 30 minutes, and incubated in donkey anti-rabbit IgG-conjugated IRDye 800CW or donkey anti-guinea pig IgG-conjugated IRDye 680RD (LI-COR, Lincoln, NE) diluted at 1:10,000 in blocking buffer for 1 hour at room temperature. The blots were washed and immediately imaged by using the LI-COR Odyssey Infrared Imaging System and evaluated by using proprietary software.