Widespread deacetylation of histone H4 occurs in apoptotic cells. Retinal sections from eyes before optic nerve crush (ONC) and at several points after ONC were double labeled with antibodies against acetylated histone H4 (AcH4, green) and γH2AX (red), to identify apoptotic cells (see Figure 5). (A) Control ganglion cell layer nuclei (N) were strongly labeled for AcH4. Arrows indicate focal γH2AX labeling associated with nucleoli (stage I). (B) Shortly after ONC, γH2AX labeling became more apparent as a perinuclear ring, principally localized to the cytoplasm (arrowhead, stage II), while nuclei of these cells still exhibited strong labeling for AcH4. (C, D, E) At subsequent time points after ONC, γH2AX labeling in nuclei (N) progressively increased (stage III), while nuclear AcH4 labeling decreased (asterisk in E). Note a change from green to yellow to red nuclear color in the merged panels. (F) By late in the cell death process, nuclei (N) intensely labeled for γH2AX exhibited little to no immunoreactivity to AcH4 (asterisk). Scale bar = 5 μm. The decrease in AcH4 labeling, and the presence of γH2AX stage III labeling, also correspond to increased DNA fragmentation as assessed by DAPI staining (See additional file 2).