Tra 1 81

To confirm pluripotency, iPSC colonies were fixed (3% paraformaldehyde × 30 min), washed with phosphate-buffered saline (PBS) and permeabilized with 0.5% Triton. After blocking with 5% BSA, the colonies were incubated overnight with primary antibodies (OCT3/4, 1:50; NANOG, 1:50; TRA-1-60, 1:200; and TRA-1-81,1:200; all from Santa Cruz, Dallas, TX) followed by secondary antibody (anti-mouse Cy3, anti-rabbit FITC, 1:1,000). Isotype control antibodies were: anti-mouse IgG2b for OCT3/4, anti-goat IgG for Sox2 and Nanog, and anti-mouse IgM for TRA-1-60 and TRA-1-81 (all Abcam, Cambridge, UK). Images were taken using a confocal laser scanning microscope (LSM 710, Carl Zeiss, Germany).
For immunocytochemistry, 4% paraformaldehyde-fixed iPSCs were incubated with 3% donkey serum in 0.3% Triton X-100 in PBS (blocking) and exposed to rat anti-human αKlotho monoclonal antibody (1:1000, KM2076, overnight at 4°C, Trans Genic, Fukuoka, Japan), followed by incubation with secondary donkey anti-rat antibody conjugated to Alexa Fluor 555 (A31570, Invitrogen, Carlsbad, CA) at room temperature × 60min. Filamentous actin was labeled with rhodamine phalloidin (R415, Invitrogen), and nuclei with 4′,6-diamidino-2-phenylindole (DAPI, P36931, Thermo Fisher, Waltham, MA), and imaged with a laser scanning microscope (Zeiss LSM 880, Advanced Imaging Microscopy, Germany).