Csu w1 spinning disk head

Third instar larvae were dissected to isolate the brain lobes and ventral nerve cord, which were placed in Schneider’s Insect media (SIM). Larval brain explants were placed in lysine-coated 35 mm cover slip dishes (WPI) containing modified minimal hemolymph-like solution (HL3.1). Treated and untreated explants were imaged on a Leica DMi8 microscope (100 × 1.4 NA oil-immersion objective) equipped with a Yokogawa CSU-W1 spinning disk head and dual Andor iXon Ultra camera. Explants expressing aPKC-GFP or Baz-GFP were illuminated with 488 nm and 561 nm laser light throughout 41 optical sections with step size of 0.5 µm and time interval of 20 s. To examine the role of F-actin in aPKC and Baz dynamics, explants were treated with 50 µM LatA (2% DMSO) during imaging.

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).

Third instar larvae were dissected in HL3.1 (Feng et al., 2004 (link)) and fixed in 4% paraformaldehyde in HL3.1 for 10 min at room temperature, then rinsed and stained with appropriate antibodies in PBS containing 0.2% (v/v) Triton X-100. Larvae were mounted in Vectashield (Vector Labs, Burlingame, CA, USA). Spinning-disk confocal Z-stacks (0.3 μm or 1 µm) were collected at room temperature on an Andor spinning-disk confocal system consisting of a Nikon Ni-E upright microscope equipped with 40× [numerical aperture (NA) 1.3], 60× (NA 1.4) and 100× (NA 1.45) oil immersion objectives, or a 60× (NA 1.0) water immersion objective, a Yokogawa CSU-W1 spinning-disk head, and an Andor iXon 897U electron-multiplying charge-coupled device camera (Andor, Belfast, Northern Ireland). Images were collected using Nikon Elements AR software and processed using Volocity software (Improvision, PerkinElmer, Waltham, MA, USA).