Anti cvh

Cells were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.5% Triton X-100 (Solarbio, Beijing, China vT8200) for 10 min. Samples were then blocked with blocking buffer (PBS containing 10% fetal bovine serum) at 37°C for 2 h or 4°C overnight. Samples were incubated with diluted primary antibodies in blocking buffer at 37°C for 2 h or 4°C overnight. After the primary antibody incubation, samples were washed three times with PBS containing 0.1% Tween 20 (Solarbio, Beijing, China, T8220) followed by the incubation with fluorescence-conjugated secondary antibodies diluted in blocking buffer for 2 h at 37°C. Then samples were washed three times with PBS containing 0.1% Tween 20. Nuclei were counterstained with DAPI (Beyotime, Beijing, China, C1002). Images were obtained using a confocal microscope (Olympus, Tokyo, Japan, FV1200). Primary antibodies included anti-CVH (Abcam, Cambridge, United Kingdom, ab13840, 1:100), anti-CKIT (Invitrogen, CA, United States, 14-1172-81, 1:100), anti-C1EIP (polyclonal antibody, 1:10), and anti-HA (Abcam, Cambridge, United Kingdom, ab187915, 1:100). Secondary antibodies included goat anti-Rat IgG (Proteintech, Chicago, United States, SA00003-11, 1:1000, [FITC] labeled) and goat anti-mouse IgG (Proteintech, Chicago, United States, SA00003-12, 1:1000, [TRITC] labeled).

Anti-H3K4me2 [ab32356; 10 μg for ChIP experiments, 1:2000 for western blotting (WB)], anti-histoneH3 (ab1791; 1:2000 for WB), goat anti-mouse IgG (ab6786), goat anti-rabbit IgG (ab6718), and rabbit anti-rat IgG (ab6730) were obtained from Abcam. Anti-Myc (14793; 1:50 for Co-IP, 1:1000 for WB) and anti-β-catenin (9587; 1:50 for Co-IP, 1:1000 for WB, 1:25 for ChIP) were obtained from Cell Signaling Technology. Anti-CVH [ab27591; 1:1000 for WB, immunohistochemistry (IHC) and fluorescence-activated cell sorting (FACS)] was obtained from Abcam.

ESCs that had been induced for 4 days were fixed with 4% paraformaldehyde for 30 min and then treated with 0.5% TritonX-100 for 15 min. After blocking with 10% fetal bovine serum at 37 °C for 2 h, the cells were incubated in anti-CVH (1:500, Abcam) for 12 h at 4 °C; After multiple washes by PBST, cells were incubated with corresponding secondary antibodies (1:400, Abcam) for 2 h at room temperature. After DAPI (5 ng/uL) staining, slides were plated with glycerin, and the image was sealed. The FV1000 laser scanning confocal microscope (Olympus, Tokyo, Japan) was used to observe the samples.