Dm5500b upright stand

The widefield microscope was a DM5500B upright stand (Leica, Germany). Acquisitions were performed using an Orca-ER camera (Hamamatsu, Japan). Mosaics were realized using a widefield/TIRF DMI6000 inverted stand microscope (Leica, Germany). Acquisitions were obtained using a DFC360 FX camera (Leica, Germany). The confocal microscope was a Nikon A1R confocal. Image acquisition was done with Leica AF suite software and analysed with image J.

Cells were fixed in 3% paraformaldehyde and permeabilized with Triton X-100. Primary antibodies included mouse anti-acetylated α-tubulin (Sigma-Aldrich, Basel, Switzerland; 1:400 dilution); rabbit anti-Arl13b (Novusbio, Abingdon, United Kingdom; 1:400 dilution). Alexa Fluor 594- and 488-conjugated secondary goat anti-mouse or goat anti-rabbit antibodies (Molecular Probes, Carlsbad, CA) were used at 1:400. Cells were visualized by wide-field, fluorescence microscopy using a DM5500B upright stand (Leica, Germany) with a 40X oil objective NA 1.00. The cubes used were A4 (excitation filter BP 360/40, dichroic mirror 400, emission filter BP 470/40), L5 (BP 480/40, 505, BP 527/30), and TX2 (BP 560/40, 595, BP645/75). Acquisitions were done with an Orca-ER camera (Hamamatsu, Japan). Cells were also visualized using the confocal microscope, Axiovert 200M inverted stand (Zeiss, Germany). Objectives 10X dry NA 0.3 and/or 25X multi immersion (oil, glycerol, water) NA 0.75, and/or 40X oil 1.3 NA and/or 63X oil 1.4 NA were used. The LASERs used were diode 405 nm, and/or Argon 488 nm, and/or HeNe 543 nm. The microscope was equipped with an automated xy stage for mosaic acquisitions.
Cilia frequency was counted manually from scans using a 40X digital zoom for 100-300 nuclei.

Cells were fixed in 3% paraformaldehyde and extracted with Triton X-100. Primary antibodies included mouse anti-acetylated tubulin (Sigma-Aldrich, Basel, Switzerland) (1:400). Alexa Fluor 594- and 488-conjugated secondary goat anti-mouse or goat anti-rabbit antibodies (Molecular Probes, Carlsbad, CA, USA) were used at 1:400. Cells were visualized by wide-field, fluorescence microscopy using a DM5500B upright stand (Leica, Wetzlar, Germany) with a 40× oil objective NA 1.00. The cubes used were A4 (excitation filter BP 360/40, dichroic mirror 400, emission filter BP 470/40), L5 (BP 480/40, 505, BP 527/30), and TX2 (BP 560/40, 595, BP645/75). Acquisitions were done with an Orca-ER camera (Hamamatsu, Hamamatsu, Japan). Cells were also visualized using the confocal microscope, Axiovert 200 M inverted stand (Zeiss, Oberkochen, Germany). Objectives 10× dry NA 0.3 and/or 25× multi immersion (oil, glycerol, water) NA 0.75, and/or 40× oil 1.3 NA and/or 63× oil 1.4 NA were used. The LASER used were diode 405 nm, and/or Argon 488 nm, and/or HeNe 543 nm. The microscope was equipped with an automated xy stage for mosaic acquisitions.
Cilia frequency was counted manually from scans using a 40× digital zoom for 100–300 nuclei.