Immunofluorescence analysis

After removing culture medium, cells attached to 12 mm round glass cover slips were fixed in ice-cold 4% paraformaldehyde in PBS on ice for 10 min. The cells were then washed three times with ice-cold PBS before incubation in 5% normal goat serum (Sigma), 0.2% saponin (Sigma) in PBS at room temperature for at least 30 min. For incubation with primary antibodies, appropriate dilutions were prepared in 5% normal goat serum, 0.2% saponin in PBS and centrifuged at 15,000 × g_max for 10 min before addition to the fixed and permabilized cells. Incubation with primary antibodies was for 60 min at room temperature, followed by three washes with PBS. Fluorochrome-coupled secondary antibodies (Invitrogen) were similarly diluted (1:1,000) in 5% normal goat serum, 0.2% saponin in PBS and centrifuged at 15,000 × g_max for 10 min before addition of aliquots of the clarified supernatant to the cells. For double transfection experiments involving both iRFP and GFP, where the efficiency of GFP expression is sharply reduced, an anti-GFP nanobody (GFP-Booster, Chromotek) conjugated to Atto 488 was used to detect GFP-expressing cells unambiguously. After incubation at room temperature for 30–45 min, the cells were washed three times with PBS, in some cases incubated briefly in 2 μg/ml Hoechst 33258 DNA dye in PBS, and mounted on glass slides in either Cytoseal (Thermo Fisher) or SlowFade Diamond (Life Technologies) mounting medium.