Immunostaining of Pax6-GFP mES Cells

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.

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Anchan R.M., Lachke S.A., Gerami-Naini B., Lindsey J., Ng N., Naber C., Nickerson M., Cavallesco R., Rowan S., Eaton J.L., Xi Q, & Maas R.L. (2014). Pax6- and Six3-Mediated Induction of Lens Cell Fate in Mouse and Human ES Cells. PLoS ONE, 9(12), e115106.

Publication 2014

ab18976 ab106465 sc-8108 ab5694

Actin Alkaline phosphatase Alpha fetoprotein Antibodies Antibody Cells Cells cultures Ectoderm Endoderm Mesoderm Neurofilament Oct4 Paraformaldehyde Smooth muscle Ssea1 Sucrose

Corresponding Organization : University of Delaware

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Variable analysis

independent variables

Pax6-GFP mES cell culture conditions

dependent variables

Expression of pluripotency markers (Oct4, SSEA1, Nanog)
Alkaline phosphatase activity
Expression of lineage-specific markers (neurofilament, alpha-fetoprotein, smooth muscle actin)

control variables

Fixation of cells in 4% paraformaldehyde (PFA)/4% sucrose
Immunostaining procedure

controls

Omission of primary or secondary antibody
Comparison of differentiated and undifferentiated cells

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