Cells were fixed with 4% paraformaldehyde containing either 0.2 or 1% Triton X-100 for 15 min at 20°C or 100% methanol at −20°C for 15 min. Fixed cells or deparaffinized slides were washed with PBS, and nonspecific binding was blocked with either 5% normal serum or 0.5% milk before incubation with primary antibodies for 1 hour at 20°C. Unbound antibody was removed by washing with PBS, and secondary antibody was bound for 30 min at 20°C or overnight at 4°C. Coverslips were then mounted with ProLong mounting media containing DAPI. Fluorescence was visualized with a Leica SP5 confocal microscope.

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