Figure 8. Nuclear localization of 5-hmC (green punctuation) after 1% DMSO treatment in MC3T3-E1 cells. The increase in 5-hmC was demonstrated and localized by immune-staining techniques. When compared with control (A), an increase in 5-hmC was already visible after 12 h of 1% DMSO treatment (B) and after 1 d (C) the effect was clearly more pronounced. Combined staining with DAPI demonstrated the signals of 5-hmCs in the nuclei and revealed that most of the hydroxymethylated spots were found outside of the heterochromatic regions (D). Co-labeling of DMSO treated cells with 5-mC (red spots) and 5-hmC (green spots) antibodies revealed areas where only 5-hmCs or 5-mCs are localized. However, also double-labeled regions were found (yellow spots, E). Scale bar for all images is 50 µm.