Ab13840

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).

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. The samples were then blocked with blocking buffer (PBS containing 10% fetal calf serum [FBS]) (Gibco, New York, USA, 10099141) at 37 °C for 2 h or 4 °C overnight. Primary antibodies diluted in blocking buffer were used for the samples and incubated at 37 °C for 2 h or 4 °C overnight. The samples were washed three times with PBS containing 0.1% Tween 20 (Solarbio, Beijing, China, T8220) followed by incubation with fluorescence-conjugated secondary antibodies diluted in blocking buffer for 2 h at 37 °C. The 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 MVH (Abcam, Cambridge, UK, ab13840, 1:100) and CKIT (Invitrogen, California, USA, 14-1172-81, 1:100). Secondary antibodies included goat anti-rat IgG (Proteintech, Chicago, USA, SA00003-11, 1:1000, FITC labeled) and goat anti-mouse IgG (Proteintech, Chicago, USA, SA00003-12, 1:1000, TRITC labeled). The directly labeled antibody was SSEA-1 (Biotechne, Minnesota, USA, IC2155T, 1:1000). PGCs were used as a positive control, and iPSCs were employed as a negative control.