HeLa cells were plated on poly-l -lysine–coated coverslips, and A459/RPE-1 cells were seeded on collagen-coated coverslips. Cells were fixed with warm 4% paraformaldehyde for 10 min at 37°C, permeabilized for 5 min in 0.1% Triton X-100, and blocked for 1 hour in 2% bovine serum albumin in phosphate-buffered saline (PBS) (blocking buffer). Coverslips were incubated in a blocking buffer containing diluted primary antibodies and incubated overnight at 4°C. Cells were washed three times for 10 min with blocking buffer and incubated with corresponding Alexa Fluorophore–conjugated secondary antibodies diluted 1:1000 in blocking buffer for 1 hour at room temperature. Cells were washed three times for 10 min with blocking buffer and once with PBS. Coverslips were mounted on a microscopic slide using fluorescence mounting medium (Dako, catalog no. S3023).
Imaging was performed using a Leica SP8 laser scanning confocal microscope and Zeiss LSM880 with AiryScan. Images of primary cilia are presented as maximum intensity projections. Image analysis was done using ImageJ. All the images were prepared for publication using Adobe Photoshop (adjusted contrast and applied 1-pixel Gaussian blur) and then assembled with Adobe Illustrator.
Imaging was performed using a Leica SP8 laser scanning confocal microscope and Zeiss LSM880 with AiryScan. Images of primary cilia are presented as maximum intensity projections. Image analysis was done using ImageJ. All the images were prepared for publication using Adobe Photoshop (adjusted contrast and applied 1-pixel Gaussian blur) and then assembled with Adobe Illustrator.