Retinal Ganglion Cell Labeling in Rotenone-Induced Retinal Degeneration

Eyes were dissected from SD rats seven days after intravitreal injection of rotenone alone or together with Ntf5 recombinant proteins and were fixed with 4% paraformaldehyde in PBS overnight at 4 °C. Fixed eyes were embedded in optimal cutting temperature compound (Sakura Finetek Japan, Tokyo, Japan) and were cut into 10 µm-thick sections using a cryostat CM3050S (Leica Biosystems, Wetzlar, Germany). Immunostaining of retinal sections was performed as reported before^{69 (link)}. Briefly, retinal sections were treated with blocking solution (10% normal donkey serum in PBS containing 0.01% Tween 20), followed by treatment with rabbit primary antibody (Abcam, Cambridge, UK; 1:500 dilutions with blocking solution) for RNA-binding protein with multiple splicing (RBPMS), which is a selective marker for retinal ganglion cells^{70 (link)}. After extensive washing, retinal sections were incubated with Cy3-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch, West Grove, PA, USA; 1:500 dilutions with blocking solution) and 4′,6-diamidino-2-phenylindole (DAPI; Dojindo, Kumamoto, Japan). To visualize the inner nuclear layer (INL) cells, retinal sections were stained with hematoxylin and eosin (HE; Muto Pure Chemicals, Tokyo, Japan).