HDAC3 becomes localized to nuclei prior to the deacetylation of histone H4 in dying cells. Retinal cryosections before and after optic nerve crush (ONC) were colabeled with either γH2AX and AcH4 or HDAC3 antibodies. Representative cells from the ganglion cell layer (GCL) are shown for four stages of γH2AX localization. (A, B) Control retinas showed nuclear labeling of acetylated H4 (AcH4) and cytoplasmic labeling of HDAC3. Representative nuclei are indicated (N). γH2AX was present as a densely labeled spot associating with the nucleoli (stage I labeling, Figure 5). (C) Early in the cell death process, nuclei (N) in the GCL were still brightly labeled for AcH4. At this stage, γH2AX had begun to appear as a perinuclear ring (arrowhead) (stage II labeling) and was not colocalized with the nuclear AcH4. Conversely, γH2AX did colocalize with HDAC3 at this stage. (D, E) These images illustrate the progressive movement of HDAC3 into the nuclei of dying cells. (D) HDAC3 was mainly present in the cytoplasm, but appeared to be concentrating around the nucleus (N). In E, HDAC3 was distributed in both the nucleus (asterisk in merged image), and colocalized with perinuclear γH2AX (arrowhead). (F, G) After HDAC3 had begun to accumulate in the nuclei, a progressive decrease in AcH4 labeling was detected. Also at this point, γH2AX was present in the affected nuclei (stage III labeling, asterisk in G). (H) HDAC3 labeling continued to increase in the nuclei and was now colocalized with nuclear γH2AX (asterisk). (I, J) At this late stage of cell death, there was virtually no AcH4 left in the γH2AX-labeled cells (asterisk in I), while γH2AX and HDAC3 remained colocalized in the nuclei (asterisk in J). A graphic representation of these changes is also shown in additional file 2. Scale bar = 2 μm.