Immunofluorescence Staining for Cytosine Modifications Like 5-Methylcytosine and Its Oxidative Derivatives and FISH

In this chapter, the protocol of combined immunofluorescence detection of 5-methylcytosine and its oxidative derivatives and FISH is given. This approach can be applied on fixed preparations of human chromosomes and nuclei. Human PHA-stimulated adult and fetal lymphocytes, uncultured tissues comprising spontaneously dividing cells, such as chorion, embryonic and testicular tissues (prepared using "direct" technique), oocytes, zygotes and blastomeres of preimplantation human embryos, and non-cultured uterine leiomyoma samples may be used. The combined immunostaining for DNA methylation allows simultaneous assessment of the whole-genome methylation pattern as well as of genomic subunits, thus indicating the functional status of nuclei with different karyotypes; it is also suited for homologous or structurally abnormal chromosomes characterizable by FISH. Combined immunostaining/FISH is an indispensable method for investigation of both programmed and abnormal de novo alterations of DNA methylation.