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 (t1/2) and mobility fractions (Mf) 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).
Ixon 897u emccd camera
Manufactured by Oxford Instruments
The IXon 897U is an Electron Multiplying Charge Coupled Device (EMCCD) camera developed by Oxford Instruments. It is designed for low-light imaging applications. The camera features a back-illuminated sensor, fast readout speeds, and a high quantum efficiency. The IXon 897U is capable of capturing high-quality images and videos in low-light conditions.
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2 protocols using ixon 897u emccd camera
1
Cilia Fluorescence Recovery Assay
2
Fluorescent Labeling of Drosophila NMJs
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Wandering 3rd instar larvae were dissected in Ca2+-free HL3.1 and fixed for 17 min in Ca2+-free HL3.1 containing 4% PFA. Larvae were blocked and permeabilized overnight in PBS containing 0.25% Saponin, 2.5% normal goat serum (NGS), 2.5% bovine serum albumin (BSA), and 0.1% sodium azide. Fixed larvae (Figure 3 - Supplement ) were stained with 1/500 FluoTag®-X4 anti-RFP (#N0404, Nanotag Biotechnologies) at 4°C for 24 hrs and with 1/100 HRP Alexa Fluor 647 at room temperature for 2hrs. Stained larvae were mounted in ProLong Diamond Antifade Mountant (#P36970; Thermo-Fisher Scientific, Waltham, MA, USA). Z-stacks were collected of Drosophila larval NMJs in muscle 4 of segment A5 with a spinning disk confocal microscope at room temperature on a Nikon Ni-E upright microscope equipped with 100x (n.a. 1.45) oil immersion objective, a Yokogawa CSU-W1 spinning-disk head, an Andor iXon 897U EMCCD camera. Images were collected using Nikon Elements AR software.
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