Sc 20109

The pathological diagnosis of tumor samples was assessed by HE and IHC staining. The primary antibodies used in IHC staining included mouse anti-pan cytokeratin monoclonal antibody (ab7753, 1:250, Abcam, Cambridge, MA, USA), rabbit anti-vimentin monoclonal antibody (ab92547, 1:250, Abcam), rabbit anti-S100 polyclonal antibody (BS1318, 1:100, Bioworld Technology, Inc. St. Louis Park, MN, USA), mouse anti-EMA monoclonal antibody (ZM-0095, 1:100, ZSGB, Beijing, China), and rabbit anti-brachyury polyclonal antibody (sc-20109, 1:250, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

The alginate beads were initially fixed in Tissue-Tek® O.C.T.™ compound (Sakura Finetek Europe, Leiden, The Netherlands) for 20 min and then snap-frozen in liquid nitrogen. Beads were then sectioned in 12 μm slices with a cryotome (Microm HM560, Thermoscientific, Reinach, Switzerland). For immune-staining, slices were incubated in solutions containing 0.1M CaCl₂ to prevent dissolution of beads. The cells were first permeabilized with 100% methanol for 2 min and then blocked with 10% FBS 0.1M CaCl₂ for 1 hour. The beads were then incubated with polyclonal rabbit brachyury (H-210) polyclonal primary antibody for 1h at a 1:200 dilution (sc-20109, Santa Cruz biotech, Santa Cruz, CA, USA). After washing, the primary antibody was visualized with the secondary antibody Alexa Fluor® 488 Goat anti-Rabbit IgG antibody (A1108, Molecular Probes, Invitrogen, Basel, Switzerland) at a 1:500 dilution. Sections were mounted in ProLong® Gold Antifade Reagent with DAPI (P-36931, Molecular Probes) to stain the nuclei and were visualized on an inverted fluorescent microscope (Nikon Eclipse E800, Nikon Instruments, Tokyo, Japan).

Protein expression of germ layer markers was investigated by fluorescent immunohistochemical staining. Teratomas were fixed overnight in 4% paraformaldehyde and embedded in O.C.T solution for cryosectioning into 10 μm sections. For immunohistochemical analysis, cryosections were permeabilized with 0.5% Triton X-100 (Sigma) for 10 min and blocked with 2% BSA (Sigma) for 1 h at room temperature. Samples were incubated overnight at 4 °C with primary antibodies (1:100 dilution) against GATA4 (sc-25310, Santa Cruz Biotechnology, Santa Cruz, CA), BRACHYURY (sc-20109, Santa Cruz), and TUJ1 (sc-58888, Santa Cruz) representing endoderm, mesoderm, and ectoderm germ layers, respectively. Primary antibody-treated samples were then washed with PBS, stained with anti-rabbit Alexa Fluor 488 (A32731, Thermo Fisher Scientific) or anti-mouse Cy3-labeled IgG (072-01-18-06, KPL, Gaithersburg, MD, USA) secondary antibodies at 1:200 dilution for 2 h at room temperature, and counterstained with DAPI (1 mg/mL; Life Technologies). The stained samples were visualized by using confocal microscopy.