Immunofluorescence Analysis of H3K9me3 and Cell Counts in Blastocysts

Zona pellucida-free embryos were washed 3 times in PBS, fixed in freshly prepared 4% paraformaldehyde in PBS, permeabilized in 1% Triton X-100 in PBS, and incubated in blocking solution (1% bovine serum albumin in PBS) for 1 h. For immunolabeling, the embryos were incubated overnight at 4 °C with an anti-H3K9me3 antibody (1:800 dilution; ab8898, Abcam) followed by 3 washes with PBS and a 1-h incubation with a FITC-labeled goat anti-rabbit IgG (1:1000 dilution; Beyotime, Shanghai, China) secondary antibody. The samples were then washed and counterstained with 4,6-diamidino-2-phenylindole (DAPI; Beyotime) to label DNA. Fluorescence was analyzed using a Nikon Eclipse Ti-S microscope equipped with a 198 Nikon DS-Ri1 digital camera (Nikon, Tokyo, Japan).
The cell numbers in blastocysts were estimated by counting nuclei stained using DAPI, and the number of TE cells was determined by counting nuclei positive for CDX2. The cell number of ICM was estimated as the total number of nuclei minus the number of TE nuclei (41 (link), 42 (link)). The blastocyst was incubated with anti-CDX2 (1:200, BioGenex Inc., San Ramon, CA) for detecting the TE, and the secondary antibody was Alexa Fluor Cy3-labeled goat anti-mouse IgG (1:1000 dilution; Beyotime, Shanghai, China).