Fig. 1. Immunological detection of 5-methylcytosine in Drosophila embryos. Embryos were double stained with an antibody that specifically recognizes 5-methylcytosine (5mC, green) and with an antibody that stains DNA (red). The left panel shows images from confocal sections of whole embryos. Scale bars: 50 µm. The right panels show an enlargement from the same embryos. Scale bars in enlarged pictures: 10 µm. (A) Early cleavage stage embryo; (B) cellular blastoderm stage embryo; (C) post-gastrulation stage embryo. (D) Pole cells from blastoderm embryos showed a distinct staining for 5-methylcytosine. (E) The specificity of the 5-methylcytosine antibody was confirmed by slot blot analysis of methylated (Bos taurus, Bt) and unmethylated (S. cerevisiae, Sc) genomic DNA. DNA from 0-6 hour Drosophila embryos (Dm) showed an intermediate staining intensity. Staining with the DNA antibody indicated equal loading of all slots. (F) 5-methylcytosine staining was found to be greatly reduced in Su(var)3-9 mutants (see text for details). (G) Incubation of dechorionated embryos with the DNA methyltransferase inhibitor 5-azacytidine reduced the 5-methylcytosine signal to background levels. The signal for DNA was not affected by the Su(var)3-9 mutation or by 5-azacytidine.