Immunostaining of Epigenetic Modifications

Embryo samples were fixed in 4% paraformaldehyde [60 (link)] for 1 h at room temperature and permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (PBS) for 30 min. To analyze 5hmC and 5mC, the samples were exposed to 2 M HCl for 15 min at room temperature and then neutralized in 0.5 M Tris-HCl (pH 8.5) for 10 min. Samples were blocked with 1% BSA and 0.2% Triton X-100 in PBS for 2 h and then incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies for 1 h at room temperature. The nuclei were stained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). Images were captured using an Olympus BX51 epifluorescence microscope (Olympus Corp, Tokyo, Japan). The primary antibodies for immunostaining included rabbit anti-5hmC (Active Motif, Carlsbad, CA, USA), mouse anti-5mC (Eurogentec, Liege, Belgium), mouse anti-H3K9me2 (Abcam, Cambridge, UK), and rabbit anti-Dppa3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA).