Alexa flour fluorescent secondary antibodies

Cells were fixed in 4% paraformaldehyde for 20 min at room
temperature and then incubated in blocking buffer (PBS containing
5%BSA and 0.2% Triton X-100) at 37 °C for 1 hr.
Cells were then incubated with primary antibodies diluted in blocking buffer
at 4 °C overnight followed by incubation with secondary antibodies
diluted in blocking buffer at 37 °C for 1 hr. Nuclei were stained
with Hoechst33342 (Invitrogen, 1:10000). Primary antibodies used include the
following: Oct4 (C-10, Santa Cruz, 1:200), Sox2 (Y-17, Santa Cruz, 1:200),
GATA4 (G-4, Santa Cruz, 1:200), Nanog (AF2729, R&D Systems, 1:200),
TUJ1 (Covance, Princeton, NJ, 1:500), Myosin (MF-20, DSHB, 1:50). Alexa
Flour fluorescent secondary antibodies (Invitrogen) were used at a 1:2000
dilution.

To induce neural differentiation, we trypsinized, collected by centrifugation, and resuspended 3 × 10⁶ to 5 × 10⁶ ESCs in 10 ml of mESC medium with 1 μM of Shield 1 (S1, Clontech, Mountain View, CA, www.clontech.com). This single-cell suspension was deposited onto a 10 cm nonadhesive bacteriological petridish. After 2 days in this condition, cells aggregated in suspension and formed embryoid bodies (EBs). Medium was changed every other day and retinoic acid (RA, sigma) was added to a final concentration of 1 μM for days 4–8. On day 9, EBs were collected and trypsinized into a single-cell suspension, which was plated into two wells of a BD Matrigel Basement Membrane Matrix (BD Biosciences)-coated 12-well plate and cultured in the mESC1S1 condition. Immunostaining was performed according to a standard protocol, using primary antibodies including Nestin (1:200, Santa Cruz) and Tuj1 (1:1000, Covance, Princeton, NJ, www.covance.com). Alexa Flour fluorescent secondary antibodies (Invitrogen) were used at a dilution of 1:2,000 and nuclei were stained with DAPI. For RT-PCR analysis, STAT3^−/− + DD-STAT3-WT and STAT3^−/− + DD-STAT3-S727A cells were collected 0, 2, 4, 6, 8, 10, and 12 days after the onset of differentiation, for RNA isolation.