Ab9582

Cultured cells were fixed in 4% PFA (Sigma-Aldrich) in PBS for 10 min and then permeabilized in PBS containing 0.3% Triton X-100 (Sigma-Aldrich). The cultures were then incubated with primary antibodies followed by secondary antibodies (see the dilutions below). 4′,6-diamidino-2-phenylindole (DAPI) (1:1,000) (Thermo Fisher Scientific) was added to visualize cell nuclei. Images were taken using the DMi8 inverted microscope (Leica). The primary antibodies used in the study were as follows: anti-HCG (ab9582, Abcam, 1:200), anti-dsRNA (MABE1134, Merck, 1:200), anti-ACE2 (ab15348, Abcam, 1:200), anti-GATA2 (WH0002624M1, Sigma-Aldrich, 1:100), anti-GATA3 (MA1-028, Invitrogen, 1:100), anti-SDC1 (12922, Cell Signaling Technology, 1:100), anti-DAB2 (ab76253, Abcam, 1:100), anti-MMP2 (40994, Cell Signaling Technology, 1:100), anti-SARS-CoV-2 nucleocapsid (MBS154642, MyBioSource, 1:300) and anti-HLA-G (ab7759, Abcam, 1:50). Secondary antibodies used in the study (all 1:400) were Alexa Fluor 488 goat anti-mouse IgG1 (A21121, Thermo Fisher Scientific), Alexa Fluor 555 goat anti-mouse IgG (A31570, Thermo Fisher Scientific), Alexa Fluor 555 goat anti-rabbit IgG (A21428, Thermo Fisher Scientific), Alexa Fluor 555 goat anti-mouse IgG2a (A21137, Thermo Fisher Scientific), Alexa Fluor 647 donkey anti-rabbit IgG (A31573, Thermo Fisher Scientific).

Conceptuses and cells growing on coverslips were fixed in 4% paraformaldehyde and were permeabilized in 0.2% triton X-100 for 10 minutes. Subsequently, conceptuses and cells were blocked at room temperature in 5% BSA in PBS for 1 hour and incubated with primary antibodies (hCGβ, Abcam, United Kingdom, ab9582; TBX3, Abcam, United Kingdom, ab99302; HLA-G, Abcam, United Kingdom, ab52454; OCT4, Santa Cruz Biotechnology, Santa Cruz, CA, sc-5279) in blocking solution overnight at 4 °C. The conceptuses and cells were then washed twice in blocking solution and incubated with species-appropriate fluorescent-conjugated secondary antibodies at room temperature for 1 hour before final washes in blocking solution. Coverslips with conceptuses or cells were then moved to drops of Vectashield mounting media with DAPI (Vector Lab, United Kingdom, H-1200) on slides for 10 minutes of incubation before imaging. For non-cocultured conceptuses, all immunostaining operations were under the stereoscopic microscope. Cell fusion index was analyzed according to previous reports [25 (link), 29 (link)].

Antibodies used for all the experiments were as follows: for immunoflourescence, β-hCG (ab9582, Abcam), Vimentin (5144 S, Cell signaling technology), E-cadherin (3195 S, Cell signaling technology), P-cadherin (sc-7893, Santa Cruz Biotechnology), BRCA1 (9010 S, Cell signaling technology) primary antibodies were followed by secondary antibodies conjugated with FITC (35552, Cell Signaling Technology). For immunoblotting, Vimentin (5144 S, Cell signaling technology), E-cadherin (3195 S, Cell signaling technology), β-actin (sc-47778, Santa Cruz Biotechnology). For immunoprecipitation: β-hCG (sc-271062, Santa Cruz Biotechnology), TGF beta Receptor II (ab78419, Abcam), normal IgG (sc-2025, Santa Cruz Biotechnology). ChIP: BRCA1 (A301-377 A, Bethyl laboratories). Immunohistochemical analysis: β-hCG (AM305-5M, Biogenex), BRCA1 (AR345-5 R, Biogenex).