Fig. 2

GFP-BAX is translocated in all compartments of retinal ganglion cells (RGCs). A A confocal image of a RGC displaying punctate GFP-BAX translocation throughout the dendritic arbor and soma (the intraretinal axon was not conclusively identified in this image) after optic nerve crush (ONC). The lower panel shows the Z-plane of the stack. Given the size and arbor stratification, this likely represents an α-ON sustained RGC. Scale Bar = 50 µm. B Retinal whole mount showing intraretinal axons filled with GFP-BAX near the optic nerve head (right). Several axons exhibit punctate localization of GFP-BAX (arrowheads). Scale bar = 20 µm. C Higher magnification of a whole mounted retina comparing diffusely labeled axons (arrows) and a punctate labeled axon (arrowheads). Punctate labeled intraretinal axons could be identified in retinas at all time points after ONC examined. Scale bar = 25 µm. D Punctate labeling of GFP-BAX in axons of the optic nerve distal to the optic nerve head. Cell death in this experiment was induced by intravitreal injection of staurosporine 24 h previously in order to preserve axon integrity with the RGC soma. An axon with diffuse GFP-BAX (arrow) is compared to an axon with punctate GFP-BAX (arrowhead). Note that the axon with punctate GFP-BAX also counterstains with an antibody against phosphorylated neurofilament (pNF) consistent with reports that degenerating neurons, including RGCs, accumulate this antigen [54, 55]. Scale bar = 4 µm