Immunofluorescence sealing solution

Spread the cells in a 6-well plate with glass slides until the cell confluence reaches 70%, fix them with 4% paraformaldehyde for 30 min, seal them with immunofluorescence sealing solution (Beyotime, P0102) at room temperature for 2 h, then incubate them with primary antibodies (Supplemental Table 2) overnight at 4 °C. On the second day, incubate with second antibody (1 : 1000) for 1 h, then stain with DAPI for 5 min, wash off with PBS, and photograph under a microscope.

The collected cloned embryos were fixed with a 4% paraformaldehyde fixative (Beyotime Biotechnology, Shanghai, China) for 15 min, washed thrice with PBS, penetrated by 0.5% TritonX-100 (Beyotime Biotechnology) for 20 min and sealed with an immunofluorescence sealing solution (Beyotime Biotechnology) at room temperature for 30 min. The embryos were incubated with CSP3-antibody (1:100; A0214; ABclonal; Wuhan, China) and goat anti-rabbit IgG H&L (1: 200; ab150077; Abcam, England) at room temperature for 1 h and at 4 ℃ for 12 h, respectively. After incubation, the embryos were washed thrice with PBS, stained with Hochest 33,342 (10 ug/mL; Yeasen Biotechnology Co., Ltd., Shanghai, China) for ten min, then washed and observed under the Nikon Eclipse Ti-s microscope (Nikon Instruments, Shanghai, China). Images were captured with the Nis Elements BR software (Nikon Instruments, Shanghai, China).