Methylation-specific fluorescence hybridization (MeFISH) originated for microscopic visualization of DNA methylation position at particular repeat sequences in specific cells. crosslinking. After denaturation fluorescent indicators were retained particularly at satellite television repeats in U-69593 wild-type however not in DNA methyltransferase triple-knockout (detrimental control) mouse embryonic stem cells. Furthermore using MeFISH we effectively detected hypomethylated satellite television repeats in cells from sufferers with immunodeficiency centromeric instability and cosmetic anomalies symptoms and 5-hydroxymethylated satellite repeats in male germ cells the latter of which had been considered to be unmethylated based on anti-5-methylcytosine antibody staining. MeFISH will be suitable for a wide range of applications in epigenetics research and medical diagnosis. INTRODUCTION DNA methylation is an important epigenetic modification of the genome in many animals and plants. In mammals it U-69593 predominantly occurs at the cytosine base of CpG dinucleotides to produce 5-methylcytosine (5mC). DNA methylation patterns are established and maintained by the members of the DNA methyltransferase family (Dnmt1 Dnmt3a and Dnmt3b) and their associated factors including Dnmt3L (1). DNA methylation plays crucial functions in the regulation of developmental gene expression chromatin remodeling genomic imprinting X-chromosome inactivation and genome stability (2). Aberrant DNA methylation is an early and fundamental event in the pathogenesis of many human diseases including cancer (3). Although the mechanism of DNA demethylation has been elusive for decades recent studies revealed that 5-hydroxymethylcytosine (5hmC) is an important intermediate for replication-dependent and/or replication-independent demethylation (4-6). A variety of methods have been developed to detect DNA methylation (7). For example the recent advancement in the high-throughput DNA sequencing technology along with the use of immunoprecipitation (8) affinity-based pull-down (9) or bisulfite conversion (10) has now made it possible to map 5mC in the genome at base resolution. At the U-69593 cellular level global DNA methylation patterns can be microscopically visualized using either anti-5mC antibodies (11 12 or methylated DNA-binding domain name fusion proteins (13 14 However methods for the microscopic visualization of 5mC in specific DNA sequences in individual cells or chromosomes have been lacking. Such FOXO3 an approach may be particularly useful for studying cells that are only available in small numbers such as early embryonic cells tissue stem cells developing germ cells and clinical specimens. It has been reported that 5mC can be distinguished from cytosine based on the large difference in osmium oxidation rate (15). Based on this chemistry a 5mC in target DNA can be detected with a DNA probe made up of a bipyridine-attached adenine derivative at the position complementary to the methylatable cytosine when treated U-69593 with osmium (16). In other words these interstrand complexes formed by osmium and nucleic acids (ICON) probes allow the sequence-selective detection of 5mC (16). In addition the ICON probes can also be used to detect 5hmC (17). In this study we applied this technology to develop a novel method named methylation-specific fluorescence hybridization (MeFISH) for visualizing the DNA methylation status at specific sequences in individual nuclei or chromosomes. MeFISH was able to detect DNA methylation at centromeric and pericentromeric repeat sequences in both mouse and human cells. Notably a high level of 5hmC at the centromeric repeats was discovered by MeFISH in developing male germ cells. We suggest that this method is suitable U-69593 for a wide range of applications in epigenetics research. MATERIALS AND METHODS ICON probes The ICON probes (Table 1) whose sequences were designed on the basis of the published satellite repeat sequences (18 19 contained a bipyridine-attached adenine derivative at the position corresponding to the methylatable cytosine (Supplementary Physique S1) (16). The probes were synthesized as described (16). In brief we created a functional nucleoside in which an adenine base and a bipyridine.