Unconjugated primary antibodies to oct3 4

Cells grown on matrigel-coated chamber slides were fixed with 4% paraformaldehyde, permeabilized with PBS containing 0.1% Triton X-100, and incubated with CAS-Block™ (Thermo Fischer Scientific Inc., Waltham, MA) for 30 minutes. Unconjugated primary antibodies to Oct3/4 (Santa Cruz Biotechnology, Dallas, Texas) and TRA-1-81 (Thermo Fischer Scientific Inc.), or fluorochrome conjugated antibodies to SOX1, Otx-2, Brachyury, and GATA4 (Human Three Germ Layer 3-Color Immunocytochemistry Kit, R&D Systems Inc, Minneapolis, MN) were incubated overnight at 4°C. When necessary, cells were incubated with fluorochrome conjugated secondary antibodies (Thermo Fischer Scientific Inc.) for 4 hrs at room temperature under humid conditions. Coverslips were mounted with fluorshield mounting medium containing nuclear counterstain DAPI (Sigma Aldrich, St. Louis, MO). Cells were visualized with confocal imaging (Nikon A1-R confocal microscope; Nikon Instruments Inc., Melville, NY).

Cells grown on matrigel-coated chamber slides were fixed with 4% paraformaldehyde, permeabilized with PBS containing 0.1% Triton X-100, and incubated with CAS-Block™ (Thermo Fischer Scientific Inc., Waltham, MA) for 30 min. Unconjugated primary antibodies to Oct3/4 (Santa Cruz Biotechnology, Dallas, Texas) and TRA-1-81 (Thermo Fischer Scientific Inc.), or fluorochrome conjugated antibodies to SOX1, Otx-2, Brachyury, and GATA4 (Human Three Germ Layer 3-Color Immunocytochemistry Kit, R&D Systems Inc., Minneapolis, MN) were incubated overnight at 4 °C. When necessary, cells were incubated with fluorochrome conjugated secondary antibodies (Thermo Fischer Scientific Inc.) for 4 h at room temperature under humid conditions. Coverslips were mounted with fluoroshield mounting medium containing nuclear counterstain DAPI (Sigma Aldrich, St. Louis, MO). Cells were visualized with confocal imaging (Nikon A1-R confocal microscope; Nikon Instruments Inc., Melville, NY).