Alexa fluor 546 conjugated transferrin

Cells were grown on coverslips and fixed 4 or 7 d postseeding for 20 min in 3.7% paraformaldehyde. Coverslips and paraffin sections were stained with primary antibodies against acetylated α-tubulin (Sigma-Aldrich), γ-tubulin (Sigma-Aldrich), EEA1 (Sigma-Aldrich), Rab11 (US Biologicals), and Alexa Fluor–conjugated secondary antibodies (Life Technologies). To study transferrin uptake, mIMCD3 cells were washed in serum-free medium and incubated in the same medium with Alexa Fluor 546–conjugated transferrin (Life Technologies) at 37°C, followed by fixation in paraformaldehyde. Images and Z-stack shots were acquired on an Olympus FluoView FV1000 confocal laser scanning microscope using an oil immersion UPLSAPO 60×/1.35 numerical aperture (Olympus, Tokyo, Japan) objective with associated software (FV10-ASW). The Z-step size was 1 μM. ImageJ software (National Institutes of Health, Bethesda, MD) was used to process images and quantify cilia. Cilia frequency was determined by counting cilia per cell. A primary cilium was considered if the long acetylated α-tubulin structure was attached to a γ-tubulin spot (acetylated α-tubulin plus γ-tubulin staining) or a long acetylated α-tubulin structure near a nucleus (acetylated α-tubulin and 4′,6-diamidino-2-phenylindole [DAPI] staining).