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Cells grown on slides were washed with 1×PBS and fixed with 4% paraformaldehyde for 20 min at 4 °C. After fixation, the cells were permeabilized with 1% Triton X-100 in 1×PBS for 20 min at 4 °C. The cells were then blocked with 5% bovine serum albumin (BSA) in 1×PBS for 1 h at 37 °C and incubated with primary antibodies as indicated overnight at 4 °C. After washing three times with 1×PBS, the cells were incubated with Alexa Fluor 594 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG at 37 °C for 1 h. Finally, the nuclei were stained by Hoechst 33342 (Sigma). Followed by washing three times with 1×PBS, the slides were visualized on a Leica DM4000 Microsystems. The fluorescent intensity spectrum of indicated single cell was analyzed by Volocity 6.3.
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