Sc 8108

For derivation of embryoid bodies (EBs), cells were cultured in pES cell medium without LIF and bFGF. The medium was refreshed daily for 10 days. Differentiation of EBs was confirmed, through RT-PCR screening for expression of markers (Table 1) associated with mesoderm (Bone Morphogenetic Protein-4, BMP-4), ectoderm (β-III tubulin) and endoderm (GATA4).
For spontaneous in vitro differentiation, day 7 EBs were mechanically dissociated and cells were plated directly onto gelatin-coated 4-well dishes in ES cell medium without inhibitors for adherent and spontaneous differentiation. After one week, cells were subjected to immunocytochemical analysis for embryonic germ layer lineages of the differentiated EB and were screened for the presence of differentiation markers: troponin I (for mesoderm; GeneTex; GTX113028), neurofilament light (NFL, for ectoderm; Millipore; AB9568), cytokeratin (for ectoderm; Sigma; C-2562), and α-fetoprotein (AFP, for endoderm; Santa Cruz; SC-8108).

Cultures of Pax6-GFP mES cells were fixed in 4% paraformaldehyde (PFA)/4% sucrose and processed for immunostaining. Commercial antibodies for Oct4 (ab18976; Abcam, Cambridge, MA), SSEA1 (FCMAB117P; Millipore, Billerica, MA) and Nanog (ab106465; Abcam, Cambridge, MA), and histochemical reagents for alkaline phosphatase activity (Sigma-Aldrich, St. Louis, MO) were used for marker studies. Differentiation of Pax6-GFP mES cells was evaluated by immunostaining with antibodies to neurofilament (NF, ectoderm) (ab24575; Abcam, Cambridge, MA), alpha-fetoprotein (AFP, endoderm) (sc-8108; Santa Cruz Biotech.) and smooth muscle actin (SMA, mesoderm) (ab5694; Abcam, Cambridge, MA). Primary and secondary antibody immunostaining were performed as previously described [38] (link). Controls included omission of primary or secondary antibody, and comparison of differentiated and undifferentiated cells. For antibody specifications, see S1 Table.

Cells or embryos were fixed with 4% paraformaldehyde fixing solution (PFA) for 15 min, washed with PBS and incubated in blocking buffer (5% goat serum, 1% BSA, 0.2% Triton-X 100 in PBS) for 45 min at room temperature. After the blocking buffer was removed, the cells were incubated overnight at 4°C with primary antibodies (5% goat serum and 1% BSA in PBS). The following antibodies were used: anti-Oct4 (1∶200, sc-5279, Santa Cruz); anti-β3 tubulin (1∶100, AT809, Beyotime); anti-α-Actin (1∶300, AA132, Beyotime); and anti-AFP (C19; 1∶100, sc-8108, Santa Cruz). On the following day, cells or embryos were washed with PBS. Afterwards, fluorescence-labelled secondary antibody (10% goat serum in PBS) 1∶300 Cy3 (A0521, Beyotime) or Alexa-594 (A21468, Invitrogen) was added and incubated in the dark at room temperature for 1 hours. The cells were washed and stained with DAPI solution (1 µg/ml).