Immunological Staining of Global Changes in DNA Methylation in the Early Mammalian Embryo

The structural complexity of chromatin can make antibody access to some nuclear antigens energetically unfavorable. This can limit the usefulness and reliability of immunostaining unless validated methods of epitope retrieval are applied. We found that denaturation of chromatin by sequential use of acidification and tryptic digestion of fixed cells is required to reliably detect DNA methylation in the embryo. Using this method to unmask the epitope revealed an unexpected pattern of reprogramming of global patterns of DNA methylation in the preimplantation embryo. This paper provides a detailed description of the procedures required for immunological detection of 5-methylcytosine in the early embryo.