Anti 5hmc antibody

Blastocysts were fixed in 4% paraformaldehyde, permeabilised using 0.5% Triton-100 in PBS, blocked in 1% BSA-PBS-0.1% PVA for 1 h at room temperature and incubated with anti-OCT4 antibody (1:400; Santa Cruz Biotechnology) and anti-CDX2 antibody (1:200; BioGenes, Berlin, Germany) overnight at 4°C. DNA was denatured with 4 M HCl for 10 min, neutralised with pH 8.0 Tris-HCl for 15 min, blocked in 1% BSA-PBS-0.1% PVA overnight and incubated with anti-5mC antibody (1:200; Abcam) and anti-5hmC antibody (1:500; Abcam) for 1 h at room temperature. After washing with PBS-0.1% PVA, the reaction was continued using Alexa Fluor 594 Goat Anti-Rabbit IgG Antibody (anti-rabbit; Invitrogen) and Alexa Fluor 488 Goat Anti-Mouse IgG Antibody (anti-mouse; Invitrogen) at a 1:1000 dilution for 1 h at room temperature. After the reaction was stopped, the embryos were washed in PBS-0.1% PVA and mounted on glass slides with mounting medium for fluorescence staining with DAPI (CA 94010, Vector Laboratories, Inc.) followed by total cell counts. Embryos were imaged immediately on an epifluorescence microscope. The number of ICM and TE cells was determined according to the number of cells positive for immunofluorescence-labelled OCT4 and CDX2, respectively. The fluorescence intensity of 5mC and 5hmC labelling was analysed using ImageJ software.