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Mouse anti 5-Methylcytidine antibody, clone 33D3 used for the detection of 5-meC in murine embryonic fibroblasts by immunofluorescence.
Staining pattern of 5meC and MBD1 antigens after epitope retrieval acid or acid plus trypsin treatment of confluent MEFs.Fixed and permeabilized MEFs were treated with HCl for 10 or 20A The proportion of nuclei (%) with focal 5meC staining (n, number of nuclei analysed). HCl (p<0.01) and trypsin (p<0.01) treatments revealed an increase in the focal staining of nuclear 5meC. 20 min HCl caused no further increase (p>0.05) after trypsin. B Typical examples of focal and diffuse staining of 5meC staining within the nuclei, yellow and red arrows indicate predominantly diffuse and focal staining, respectively. Non-immune IgG control staining is also shown. C The total level of 5meC staining of treatments shown in A. The results are the mean ± SEM of arbitrary units of optical density increased (**** p<0.0001). D The average number of 5meC foci per cell in A. E The average size (cross-sectional area) of 5meC foci in cells in A. F Representative images of MBD1 staining after 10 min HCl and trypsin 0, 1 or 2 min. Blue and red arrows indicate large and small foci of MBD1, respectively, and yellow arrow shows diffuse staining of MBD1. G The proportion of nuclei with focal MBD1 staining increased after 1 min trypsin (p<0.01). H The total level of MBD1 staining (arbitrary units of optical density) increased after 10 min HCl and 1 min trypsin (p<0.0001) Mean +/- standard mean of error. Results are from three independent replicates. ** p<0.01 and **** p<0.0001. Scale bar 10 µm. If not shown on individual graphs, the relevant statistical outcomes are stated within the results text.
From: Çelik S, Li Y, O'Neill C (2014) The Exit of Mouse Embryonic Fibroblasts from the Cell-Cycle Changes the Nature of Solvent Exposure of the 5'-Methylcytosine Epitope within Chromatin. PLoS ONE 9(4): e92523.