Figure 2
Specificity and sensitivity of used anti-methylcytosine antibody. (A) The antibody properties were assessed by an indirect immunofluorescence assay, in which the monoclonal anti-MeC antibody for this study — used at the concentration of 1 μg/ml in combination with a secondary antibody (Cy3-conjugated anti-mouse IgG1 at 5 μg/ml), i.e. at the same concentrations as in the cellular assay — was hybridized to a spotted array with two types of short 24-mer oligonucleotides immobilized onto a glass slide: C-oligo that included two CG dinucleotides and its methylated counterpart, the MeC-oligo printed at various dilutions that correlate with different approximate CpG copy numbers (1010–104) . Each DNA probe was spotted as octuple. The specific antibody, detected with a microarray scanner at 5 microns resolution, shows best signal-to-noise (background and non-specific binding to unmethylated C-oligo) ratio at a copy number of 1010. The signal (false-colored in green) decreases in a CpG copy number-dependent manner. (B) Similar average intensities were obtained, when a sub-area (magenta box in Figure2A) of the same array was subjected to confocal scanning microscopy at 200 nm horizontal resolution. The line scan (magenta) shows the more detailed intensity profile across the four different types of spots and the intermediate gaps (coated glass slide/background).