All steps were performed at 18–24°C. Cells were fixed with 4% paraformaldehyde in phosphate‐buffered saline (PBS) for 20 minutes, permeabilized with 0.2% Triton X‐100 containing PBS, then blocked in 3% goat serum, then incubated with appropriate primary and secondary antibodies. Nuclei were counterstained with either DAPI (4′,6‐diamidino‐2‐phenylindole, Sigma‐Aldrich) or Hoechst staining (bisbenzimide; Sigma), and coverslips were mounted on slides with FluorSave (Merck, NJ, USA). Fluorescent imaging was performed using an Axioscope (Zeiss, Oberkochen, Germany) microscope. Images were processed with Axiovision V 4.8.1 (Zeiss). Regions of interest were selected based on uniform DAPI staining and imaged for two to four fluorescent channels, and immunolabeled cells counted manually with ImageJ64 (v 1.47) software for an area of 162.40 μm2. Positive staining was defined as a signal clearly above the background fluorescence present in areas devoid of cells. At least three independent images per culture and at least three independent cultures from three independent conversions were analyzed.
