Figure 2. The effect of antigen retrieval procedures on the detection of 5hmC and 5meC in the mouse PN5 stage zygote.

Zygotes were collected directly from the oviduct and fixed and prepared for staining. After antigen retrieval steps, zygotes were incubated with primary antibodies for 1 h at room temperature (5hmC) or 18 h at 4°C (5meC) and each primary antibody was detected by an FITC-labeled secondary antibody. The pronuclei were counter-stained for DNA with PI and the FITC and DNA images merged. Three epitope retrieval approaches were under taken: −H-T, zygotes were not pretreated with either acid (HCI) or trypsin; +H-T, they were pretreated with acid only; and +H+T, epitope retrieval was by a combination of acid and trypsin treatment. The images are representative of three independent replicates. In each case conditions for staining and imaging were managed so that no signal was detected from the non-immune negative control treatments, and these same conditions used for imaging primary antibody staining. The bar is 5 µm and all images are at the same magnification.