Understanding the complexity of antigen retrieval of DNA methylation for immunofluorescence-based measurement and an approach to challenge.

Cytosine methylation (5-methylcytosine, 5meC) in the CpG-rich regions of the mammalian genome is an important epigenetic mechanism playing roles in transcription regulation and genomic stability. The abnormalities in DNA methylation can occur in various types of cancer and some genetic diseases. The measurement of DNA methylation is therefore important and there is a range of methodologies used to detect DNA methylation. Many methods based on bisulfite treatment appeared with a lack of specificity after recent discoveries of various modifications of methylated cytosine, however there are new treatments developed to overcome this limitation. Immunofluorescence is currently known to be able to specifically detect DNA methylation as it uses different antibodies against 5meC and its derivatives, but it is a semi-quantitative method. Immunofluorescence protocols commonly include fixation of cells followed by permeabilisation, antigen retrieval, and treatments with antibodies. Establishing the strategy for antigen retrieval of immunofluorescence is important to unmask epitopes (i.e. 5meC) from other proteins, and therefore to access the antigen of interest. There are many approaches used for antigen retrieval induced by acid, enzyme and/or heat. The selection of antigen retrieval method can depend on a variety of such antigen-based or cell-based conditions, since the dynamic structure of DNA and chromatin accounts for the complexity of involved proteins to mask the epitope. This review aims to specifically focus on the complexity of in situ detection of DNA methylation by immunofluorescence-based methods using antigen retrieval with the current understanding of DNA methylation mechanism, and suggests conditions for antigenic retrieval of 5meC epitope.