Wave fx spinning disc confocal microscope

In six-well plates, coverslips were precoated with 50 µg/ml of poly-d-lysine (Sigma, Markham, ON, Canada) and 700,000 RB cells and 300,000 dissociated RPC were seeded in 2 ml/well overnight. Where applicable, cells were labeled with MitoTracker Deep Red FM (Cell Signaling, Whitby, ON, Canada) or EdU as per manufacturer’s instructions. Cells were fixed with 4% paraformaldehyde for 10–15 min, permeabilized and blocked for 1 h, then subjected to click chemistry and probed with primary antibodies overnight at 4 °C as in Table S3. After 2–3 washes, cells were probed with Alexa Fluor 488- or 568-conjugated secondary antibodies (ThermoFisher, Burlington, ON, Canada) and 4,6-diamidino-2-phenylindole or propidium iodide (PI) for 90 min. Coverslips were mounted using VectaShield (Vector Laboratories, Burlington, ON, Canada). High resolution confocal images were acquired with the Wave FX Spinning Disc Confocal microscope (Quorum Technologies, Puslinch, ON, Canada) and Volocity software.

Worms were anesthetized on 25 mM of sodium azide in M9 buffer and mounted on 2% agarose pads for live imaging. Images are projections of 3D stacks collected using a Delta Vision (GE) deconvolution system (0.2 μm optical sections) or a WaveFX spinning disc confocal microscope (Quorum Technologies). Deconvolution, when necessary, was performed using Softworxs (GE). Expansion of TAC-1::GFP domain into the TZ and axoneme was quantified using the line profile tool in Softworks. Relative fluorescence intensities collected from phasmid cilia using the same acquisition parameters were measured in N2 (n = 10) and sgo-1(tm2443) (n = 14) worms expressing MKS-5::mCherry and TAC-1::GFP across a 2 mm transect traced from the base of the TZ (boundary between the MKS-5::mCherry (TZ) and TAC-1::GFP (BB) domains) along the axoneme distally. Images of worms expressing Pcol-183::mCherry were taken on a Ds-Fi1 camera mounted on a Nikon SMZ1500 fluorescent stereomicroscope.