Immunofluorescence Imaging of Cilia

Cells (50 × 10⁵ cells per well) were plated on glass coverslips in a six-well plate for 18 hours, fixed, and permeabilized using 4% paraformaldehyde (20 min) and 0.1% Triton X-100 in 1× PBS (pH 7.4) for 10 min. The cells were then blocked with 1% bovine serum albumin and dissolved in PBS (pH 7.4) for 1 hour. Cells were incubated for 18 hours at 4°C with primary antibodies in blocking solution followed by Alexa Fluor 488–, Alexa Fluor 594–, or Cy5-conjugated secondary antibodies (1:500) for 60 min. Immunofluorescence was performed using a Leica TSC SP2 AOBS TCS confocal or Olympus FV10i microscope with 543-nm and 488-nm channels for visualizing red and green fluorescence (50 (link)). Cilia were imaged using anti–Ac-tubulin antibody. Images were taken at 63× magnification. At least three random fields were selected for images. Images and overlays were analyzed in Leica LAS AF software (52 (link), 68 (link)).