Elements ar software

Formalin-fixed, paraffin-embedded tissue was used for analyses. Sections were cut to 5 mm, deparaffinized, and stained with haematoxylin and eosin (Sigma-Aldrich) or processed for immunofluorescence. Samples were deparaffinized in xylene, rehydrated in graded alcohol, and rinsed in PBS, followed by antigen retrieval with boiling citrate buffer, pH 6. Slides were blocked in TBST (1× TBS and 0.05 % Tween 20) with 10 % goat serum. Anti-human mitochondrial antibody (Abcam ab92824, 1:800 dilution) was diluted in TBST and 1% BSA, and slides were incubated overnight in a humidified chamber at 4°C. Slides were washed in TBST prior to application of secondary antibodies of Alexa Fluor 488-conjugated anti-mouse antibodies (Cell Signaling #4408, 1:1000 dilution) in the dark for 1 h at room temperature in a humidified chamber. Slides were then washed in TBST and the coverslips mounted with ProLong Gold antifade reagent with DAPI (Life Technologies) for nuclear counterstaining. haematoxylin and eosin stained slides were imaged using a Nikon E600 microscope and photographed with a Nikon CCD digital camera using Elements AR software (Nikon), and immunofluorescence was visualized using a Olympus IX81 motorized inverted microscope and photographed with a Hamamatsu Photonics C9100-02 EMCCD camera using Slidebook software (3i).

Animals were mounted on 10% agarose pads and immobilized using 10 mM tetramisole. Animals were imaged at 0.6 μm interval z-stacks using a 100X oil immersion objective on an upright spinning disk microscope (Nikon Ni-E with a Yokogawa CSU-W1 spinning disk head and an Andor iXon 897U EMCCD camera). Images were collected using Nikon Elements AR software. Cilia were photobleached using a 405 nm laser (at 40% power), directed by an Andor Mosaic three digital micromirror device. One or both AWB cilia were photobleached in wild-type and odr-1 mutants. Cilia were imaged at least 12 s prior to bleaching, and up to 2 min following the bleaching event at 3 s intervals to assess fluorescence recovery. Images were corrected for photobleaching using the Bleach Correction plugin and Simple Ratio Method in FIJI/Image J [National Institutes of health (NIH), Bethesda, MD]. Pre-bleach fluorescence was normalized to 100% in order to calculate the fraction of fluorescence recovery. The recovery half-times (t_1/2) and mobility fractions (M_f) were calculated using Prism 6 Software (Graphpad, La Jolla, CA) by fitting individual recovery curves using one phase association nonlinear regression. The mean fluorescence recovery curves were created by plotting the mean and SEM of fluorescence intensities at individual time points after bleaching using Prism 6 Software (Graphpad, La Jolla, CA).