Anti human ssea4

The cloned cells (1 × 10⁶ cells/mL) were analyzed by FCM. Either anti-human SSEA-4 (an iPS cytoplasmic protein marker) mouse monoclonal antibody or anti-human TRA-1-60 (an iPS cytoplasmic protein marker) mouse monoclonal (IgM) antibody (1:100; Abcam plc., Cambridge, United Kingdom) was added to the cells as the primary antibody and incubated at 4°C for 20 minutes. As secondary antibody, Alexa Fluor 488 labeled anti-mouse IgG goat monoclonal antibody, or Alexa Fluor 488 labeled anti-mouse IgM goat monoclonal antibody (1:500; Thermo Fisher Scientific, Inc.) was added and incubated at 4°C for 20 minutes. An FACScan (Becton Dickinson) was used for cytometric analysis. To remove the dead cells from the sorting, 1 μg/mL of propidium iodide (Thermo Fisher Scientific, Inc.) was added to the samples.

Cell lines were rinsed with 1X PBS, fixed with 4 % paraformaldehyde (Santa Cruz, #sc-281692) in PBS for 5 min at room temperature. Nonspecific binding sites were blocked by incubation with PBST (PBS supplemented with 0.1 % Triton X-100) containing 10 % donkey serum (Jackson Labs, #017-000-1210) for 30 min at room temperature. Cells were subsequently incubated overnight at 4 °C in PBST containing 10 % donkey serum and specific primary antibodies: 1:500 anti-Human OCT4 (Stemgent, #09-0023), 1:100 anti-human NANOG (Cell Signaling Technologies, #4903), 1:250 anti-human SSEA4 (Abcam, #ab16287), 1:500 anti-TRA-1-60 (Life Technologies), 1:200 anti cTnT, 1:200 anti-ACTIN, 1:200 anti-SOX1, 1:200 anti-SOX2, 1:200 anti-NESTIN, 8 ng/ml anti-SOX17, 10 ng/ml anti-ALB, 1:500 anti-Sendai Vector (MBL, #PD029). Following a 3 times wash with PBS, cells were incubated with one of the following secondary antibodies: Alexa Fluor® 488 donkey anti-Mouse (#A-21202; 1:1000 dilution), Alexa Fluor® 555 donkey anti-goat IgG and Alexa Fluor® 555 donkey anti-rabbit IgG (#A-21428; 1:1000 dilution). After washing 3 times with PBS, the samples were incubated for 10 min with Hoechst (1 μg/ml) in PBS, followed by a final wash in PBS. Fluorescence images were captured with the Celigo, Nikon Eclipse TE 2000-U or Olympus BX41 fluorescent microscopes.

For cell staining, cells on the coverslip were washed with PBS and fixed in 4% paraformaldehyde. For tissue staining, the whole cornea with limbus was carefully harvested and embedded in OCT compound. Eight-μm frozen sections were made and fixed in cold acetone, after which specimens were treated with 0.1% Triton X-100 for 30 min and blocked with 5% BSA for 1 h. Fluorescein conjugated or non-conjugated primary antibody at appropriate concentration was used for incubation at 4°C overnight. If necessary, specimens were then incubated with fluorescent second antibody at room temperature for 2 h. Finally, after stained with DAPI for 5 min, slides were mounted with mounting medium and observed with a fluorescence microscopy (Nikon, Tokyo, Japan). The primary antibodies used were as follows: anti-human p63, anti-human Ki67, anti-human ABCG2, anti-human CK3/12, anti-human ABCB5, anti-human SSEA4, anti-human Nanog, anti-human OCT4 (all from Abcam, Cambridge, MA), anti-rabbit CD11b, anti-rabbit CD161, anti-rabbit CD4, and anti-rabbit CD8 (all from Biolegend, San Diego, CA).