Immunofluorescence Imaging of Cell Cultures

Immunofluorescence analysis was done as we previously described [50 (link), 51 (link)]. Cells were fixed in 100% methanol for 15 min at room temperature. For staining, samples were permeabilized for 15 min in freshly prepared PBS containing 0.25% Triton-X100, then blocked for 1h in 5% donkey serum, 0.1% fish gelatin, 0.2% Tween-20 and PBS. Samples were then incubated in 37°C water bath for 1 h with 1 mg/ml of primary antibody diluted in blocking solution. Samples were transferred to a 1:500 dilution of Goat anti-mouse IgG Rhodamine (Thermo Fisher Scientific, Waltham, MA) or Goat anti-rabbit IgG Fluorescein in blocking solution and incubated for in 37°C water bath for 1 h. Cells 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).