X1 disk

Dye-filling assays were performed as described previously with modifications (PERKINS et al. 1986) . Briefly, synchronized L4 animals were washed off NGM plates using M9 buffer and collected into 1.5 ml tubes. Following two washes with M9 buffer, animals were resuspended in 200 µl of M9 and 1 µl of 2 mg/ml DiI (Molecular Probes, Carlsbad, CA) in dimethylformamide (DMF) was added. Animals were incubated in DiI in M9 for 2 hours, rocking gently. After incubation, animals were washed twice with M9 buffer and returned to a fresh NGM plate with OP50. Dye-filling in amphid and phasmid neurons was observed in adults 24 hours later using a Nikon SMZ18 fluorescence stereomicroscope. Worms were scored as "Normal" if all amphid/phasmid neurons showed complete dye-filling, "Partial Dyf" if some dye-filling was lost in the amphids/phasmids, and "Dyf" if there was no dye-filling detected in the amphids/phasmids.
For fluorescent imaging, animals were anesthetized using 10 mM levamisole in M9 and immobilized on a 2% agar pad. Confocal images were captured on a Nikon Spinning-disk confocal microscope with Yokogawa X1 disk, using Hamamatsu flash4 sCMOS camera. 60x apo-TIRF (NA=1.49). Images were processed using Nikon Elements and ImageJ software.

Ten (10) µm thick tissue cryosections were used for immunofluorescence microscopy. Sections were fixed with 4% PFA for 10 minutes, permeabilized with 0.1% Triton X-100 in PBS for 8 minutes and then blocked in a PBS solution containing 1% BSA, 0.3% TritonX-100, 2% (vol/vol) normal donkey serum and 0.02% sodium azide for one hour at room temperature. Primary antibody incubation was performed in blocking solution overnight at 4°C. Primary antibodies include antiacetylated α-tubulin (Sigma, T7451) direct conjugated to Alexa 647 (Invitrogen, A20186) and used at 1:1000, anti-Arl13b (Proteintech, 1771-1AP, 1:500). Cryosections were then washed with PBS three times for five minutes at room temperature. Secondary antibodies, donkey anti-rabbit conjugated Alexa Fluor 594 (Invitrogen, 1:1000), diluted in blocking solution were added for one hour at room temperature. Samples were washed in PBS and stained with Hoechst 33258 (Sigma-Aldrich) for five minutes at room temperature. Cover slips were mounted using Immu-Mount (Thermo Fisher Scientific). Fluorescence images were captured on Nikon Spinning-disk confocal microscope with Yokogawa X1 disk, using a Hamamatsu flash4 sCMOS camera. 60x apo-TIRF (NA=1.49) or 20x Plan Flour Multi-immersion (NA=0.8) objectives were used. Images were processed using Nikon's Elements or Fiji software.