Localization of γH2AX in apoptotic retinal ganglion cells (RGCs). Three stages of γH2AX localization in the ganglion cell layer (GCL) were apparent. (A) γH2AX labeling in healthy cells (Stage I) was detectable as a prominent spot associated with the nucleolus (arrow). Stage II labeling was characterized by a cytoplasmic perinuclear ring. Strong nuclear labeling (N) was indicative of stage III γH2AX localization. Scale bar = 3 μm. (B) Quantification of γH2AX labeling in control and crush retinas. In control eyes (day 0), the majority of cells in the GCL exhibited stage I labeling with a small number of cells showing stage III labeling. As early as 1 day post ONC, stage II labeling was present in ~40% of cells in the GCL with a small number of stage III labeled cells. Stage III labeling increased through days 3, 5, and 7 post ONC. No more than 50% of cells in the GCL exhibited stage II or III γH2AX labeling, suggesting that a subset of cells were affected. (C) To verify that the subset of γH2AX labeled cells were RGCs, they were prelabeled with Fluorogold (FG) prior to ONC and staining for γH2AX (5 days post ONC). The images show 3 FG positive cells in the GCL with stage II γH2AX labeling (arrows), while a fourth FG labeled cell (asterisk) shows relatively weak γH2AX labeling. In the control eyes, < 15% of FG-positive RGCs exhibited stage II or III γH2AX labeling versus 85% of FG-positive RGCs in experimental eyes. Scale bar = 10 μm.