Mab1368

Differentiated cells from embryoid bodies were fixed on dishes with 4% PFA overnight at 4°C, then washed 3 times with PBS(-) for 5 min. Expression of α-SMA, β-tubulin or AFP in cultured cells was detected with the indicated antibody: anti-α-SMA antibody (1:200; V6630, Sigma, Tokyo, Japan); anti-β-tubulin antibody (1:100 T4026; SIGMA); anti-AFP antibody (1:100 MAB1368; R&D Systems, MN, USA). Binding was visualized with a secondary antibody labeled with Alexa Fluor 488 (1:500; A11008, Life Technologies) or Alexa Fluor 594 (1:500; A11062, Life Technologies), and cells were counterstained with DAPI (D1306, Life Technologies). An Olympus IX71 fluorescence microscope was used for fluorescent observation.

For immunostainings, samples (mouse ESCs and EBs) were fixed in 4% PFA for 10 min at rom temperature, washed with PBS, and permeabilized in 0.3% triton X-100 in PBS for 10 min at room temperature. Primary antibodies used were anti-Oct4 (Abcam, ab18976), anti-Nanog (Novus Biologicals, NB100-58842), anti-Sox2 (Abcam, ab97959), anti-SSEA1 (Cell signaling Technologies, MC480), anti-Nestin (Abcam, ab11306), anti-Smooth Muscle Antibody (Abcam, ab5694), anti-Alpha-fetoprotein (R&D System, MAB1368). After overnight primary antibody incubation, samples were washed with PBS and incubated with secondary Alexa Fluor^®488-coniugated antibodies (Life technologies), diluted 1:2000. Samples were also counterstained with DAPI, 200 μg/ml. Slides were observed using an Olympus BX61 Research Microscope equipped with a cooled CCD camera. Images were captured and analyzed with Applied Imaging Software CytoVision.

Porcine iPS cells were cultured in a 35 mm Petri dish through suspension culture (2 × 10⁶ cells/plate) in 2i medium without Dox. The culture medium was replaced every 2 days. After 5 days in suspension culture, the formed embryoid bodies (EBs) were transferred to a gelatin-coated culture dish allowing the spontaneous differentiation for another 5 days. The cells were then fixed and used for immunocytochemistry analysis to detect markers of the three germ layers, including TUJ1 (MMS-435P, Covance, USA) for ectoderm, DESMIN (MAB3430, Millipore) for mesoderm, and AFP (MAB1368, R&D System, USA) for endoderm.

For the immunocytochemical determination of pluripotency and differentiation markers, cells were fixed with PBS containing 4% (vol per vol) paraformaldehyde for 10 min at room temperature. The cells were permeabilized with PBS containing 0.1% Triton X-100 for 10 min at room temperature, then washed with PBS and treated with 1% BSA for blocking. For hiPSCs or reprogramming HDFs, the primary antibodies used were SSEA4 (0.5 µg per mL; eBiosciences), TRA-1-60 (0.5 µg per mL; eBiosciences), NANOG (2 µg per mL, AF1997; R&D Systems), OCT3/4 (1:200, sc-5279; Santa Cruz Biotechnology), MAP2 (1:1000, AB5622; Millipore), α-SMA (1:1000, A2547; Sigma), and AFP (2 µg per mL, MAB1368; R&D Systems). The secondary antibodies used were Alexa Fluor 488- or 555-conjugated goat anti-mouse IgG (1:200; Invitrogen), Alexa 488- or 555-conjugated goat anti-rabbit IgG (1:200; Invitrogen), and Alexa 488- or 555-conjugated donkey anti-goat IgG (1:200; Invitrogen). Nuclei were stained with the 4’,6-diamidino-2-phenylindole (DAPI) contained in the VectaShield set (Vector Laboratories). The samples were analyzed in randomly selected images with BZ-X710 (Keyence, Osaka, Japan).

Human iPSCs and iPSC‐derived neural cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature (RT). Prior to immunocytochemistry, cells were permeabilized with 0.1% Triton/phosphate‐buffered saline (PBS) for 15 min at RT and blocked with either 10% goat serum/PBS or 10% horse serum/PBS. Primary antibodies used were as follows: OCT4 (1:100; Santa Cruz, sc‐5279), NANOG (1:100; Abcam, AB80892), Tra‐1‐60 (1:100; Santa Cruz, sc‐21705), SSEA4 (1:100; BD Bioscience, 560796), α‐fetoprotein (1:150; R&D Systems, MAB1368), α‐smooth muscle actin (1:150; R&D Systems, MAB1420), Sox1 (1:100; R&D Systems, AF3369), Sox2 (1:100; R&D Systems, 245610), Nestin (1:500; Abcam, ab22035), Ki67 (1:100; BD Pharmingen, 550609), OTX2 (1:250; Millipore, AB9566), Pax6 (1:300; Biolegend, 901301), vimentin (1:100; Abcam, ab28028), vGLUT1 (1:2000; Synaptic systems, 135303), and β‐III tubulin (Tuj1) (1:150; R&D System, MAB1195). All primary antibodies were diluted in blocking solution and incubated overnight at 4°C, followed by washing in PBS 3 times for 15 min. Subsequently, appropriate Alexa Fluor 488 or 564 conjugated secondary antibodies were incubated for 1 h at RT. For nuclei staining, samples were incubated with DAPI (Sigma‐Aldrich) for 5 min and washed in PBS briefly. Finally, images were captured and analyzed using IN Cell 2200 (GE Healthcare).

Alkaline phosphatase staining was performed using the Vector Alkaline Phosphatase Substrate kit (Vector). For immunofluorescence analysis, cells were fixed with PBS containing 3.7% paraformaldehyde for 10 min at room temperature. After washing with PBS, cells were blocked with 5% bovine serum albumin (Sigma) and 0.1% Triton X-100 (Sigma) for 45 min at room temperature, and then incubated overnight at 4°C with primary antibodies against OCT3/4 (1:25, SC-5279, Santa Cruz Biotechnology), NANOG (1:250, AB5731, Millipore), glial fibrillary acidic protein (GFAP, 1:100, Z0334, DAKO), actin smooth muscle (ASM, 1:1000, MS-113-P0, Thermo), or alpha-fetoprotein (AFP, 1:100, MAB1368, R&D Systems). Alexa Fluor 594 goat anti-mouse IgG or IgM (1:500, Invitrogen), Alexa Fluor 594 goat anti-rabbit IgG (1:500, Invitrogen), Alexa Fluor 488 goat anti-mouse IgG or IgM (1:500, Invitrogen), and Alexa Fluor 488 goat anti-rabbit IgG (1:500, Invitrogen) were used as secondary antibodies. Nuclei were stained with 1 μg ⁄mL Hoechst 33342 (Sigma).

To examine the pluripotency of the PBMC-hiPSCs, immunocytochemistry was performed as described previously (Yamasaki et al.2014 (link)). Briefly, the cells fixed with 4% paraformaldehyde were stained with antibodies against Oct4 (MAB4401, mouse monoclonal, 1/200, Millipore), Tra-1-60 (09-0010, mouse monoclonal, 1/200, Stemgent®, Cambridge, MA), and SSEA-4 (MC 813-70, mouse monoclonal, 1/100, R&D Systems Minneapolis, MN), and differentiated cells were stained with antibodies against βIII-tubulin (MAB3408/1637, mouse monoclonal, 1/300, Chemicon, Burlington, MA), α-smooth muscle actin (N1584, mouse monoclonal, pre-diluted, DAKO Cytomation, Glostrup, Denmark), and α-fetoprotein (MAB1368, mouse monoclonal, 1/100, R&D Systems) (Table 1). These primary antibodies were visualized with Alexa Fluor® 488-conjugated goat anti-mouse IgG (A11001, 1/300, Invitrogen, Carlsbad, CA). The cell nuclei and double-stranded DNA were stained with 4′, 6-diamidine-2′-phenylindole dihydrochloride (DAPI). Fluorescence images were acquired using a Zeiss inverted LSM 700 confocal microscope (Carl Zeiss, GmbH, Oberkochen, Germany).