Ab106465

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.

The following antibodies were used: anti-Bnip3 polyclonal antibody (3769, Cell Signaling Technology), anti-Actin monoclonal antibody (A5441, Sigma Aldrich), anti-LC3B antibody (PM036, Medical and Biological Laboratories, Co.), anti-Sox2 monoclonal antibody (ab92494, Abcam, for western blotting), anti-Sox2 polyclonal antibody (AF2018, R&D, for IF), anti-Oct4 polyclonal antibody (ab19857, Abcam), anti-Nanog polyclonal antibody (ab106465, Abcam), anti-γH2AX polyclonal antibody (CST7918, Cell Signaling Technology), anti-53BP1 polyclonal antibody (NB100-304, Bio Techne), anti-p-ATM (Ser1981) monoclonal antibody (CST13050, Cell Signaling Technology), anti-ATM monoclonal antibody (CST2873, Cell Signaling Technology), anti-p-p53(Ser15) monoclonal antibody (CST9284, Cell Signaling Technology), anti-p53 monoclonal antibody (CST2527, Cell Signaling Technology), anti-Rad51 monoclonal antibody (H00005888, Abnova), anti-p-AMPK(Thr172) (CST2531, Cell Signaling Technology), anti-p-AMPK (CST2532, Cell Signaling Technology), anti-SSEA-1 monoclonal antibody (SC-21702AF647, Santa Cruz Biotechnology), Alexa Fluor 488 donkey anti-rabbit IgG (A21206, Invitrogen Thermo Fisher Scientific), Alexa Fluor 594 donkey anti-mouse IgG (A 21203, Invitrogen Thermo Fisher Scientific), Alexa Fluor 647 donkey anti-goat IgG (A32849, Invitrogen Thermo Fisher Scientific).

Mouse embryonic stem (ES) cells were used for recellularization. Fluorescence analysis was performed to identify the property of stem cells. Briefly, cells were washed once with PBS, and fixed using 4% PFA in 1X PBS. After fixation, cells were blocked and permeabilized for 1 h at 37°C with 5% goat serum/1X PBS with 0.1% Triton X-100. Subsequently, cells were incubated with the indicated primary antibody overnight at 4°C. The cells were then washed three times with 1X PBS and incubated for 1 h at 37°C with the respective secondary antibodies. 4,6-Diamino-2-phenylindole (DAPI) was used for nuclear counterstaining. Cells were washed three times with 1 ×PBS before analysis. The primary antibodies included rabbit polyclonal to Nanog (Abcam, ab106465), rabbit polyclonal to SOX2 (Abcam, ab97959), rabbit polyclonal to OCT4 (Abcam, ab18976), rabbit monoclonal to pax-2 (Abcam, ab79389). The second antibody was FITC-conjugated anti-rabbit antibodies (Zhongshan Goldenbridge, China)