Cells were fixed in 100% methanol for 15 min at room temperature. For staining, samples were permeabilized for 15 min in freshly prepared IF staining solution (PBS containing 0.02% saponin, 1% bovine serum albumin, and 0.05% sodium azide, 0.2% Triton X-100). Samples were then incubated in a 37°C water bath for 1h with a primary antibody diluted in IF solution. Antibodies in ES characterization kit were purchased from EMD-Millipore Chemicals (Billerica, MA) and used in 1:50 dilution as recommended by the manufacturer. The same isotype Ig was used as background control. Samples were transferred to a 1:500 dilution of goat anti-mouse IgG rhodamine (Thermo Scientific, Waltham, MA) in IF solution and incubated for in 37°C water bath for 1h. Processed samples were mounted on a glass slide with mounting medium with DAPI (Vectashield, Burlingame, CA) and visualized with an inverted light microscope (Olympus IX81 and CellSens Dimension software, Center Valley, PA).
