Anti ssea 3

Cells previously fixed for 15 min at room temperature in Phosphate-Buffered Saline (PBS) with 4% paraformaldehyde were blocked with 0.1% Triton-X, 5% donkey serum, and 1% bovine serum albumin in PBS. The following primary antibodies were used in this study: SOX2 (1 : 100, Millipore), FOXG1, PAX2, HATH1 (ATOH1) (all 1 : 100, Abcam UK), PAX8 (1 : 100, Santa Cruz), POU4F3 (BRN3C, 1 : 50, Abnova), POU4F1 (BRN3A, 1 : 100, Chemicon), and B-tubulin III (1 : 100, Sigma). Secondary antibodies used were anti-mouse, anti-goat, or anti-rabbit Alexa Fluor 488 and 568 (Molecular Probes, Life Technologies, UK), while nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma). Cells were imaged either on an EVOS FL Cell Imaging System or using the IN Cell Analyzer 2000 system platforms (GE Healthcare). Quantitative immunofluorescence was performed on the IN Cell Analyzer using the Developer Toolbox. Approximately 100-200 fields per antibody staining condition and per cell line were analyzed, capturing between 1,400 and 15,000 cells per condition, per line. Statistical comparison across the different antibody conditions and reprogramming methods was done using 2-way ANOVA. Results are reported as mean% ± SEM. The anti-SSEA3, SSEA4, TRA-1-60, and TRA-1-81 antibodies used for flow cytometry were from BioLegend.