To study microtubule polarity in developing neurons, the 1407-Gal4, UAS-EB1-GFP; UAS-dicer2 tester line was crossed to control or Patronin RNAi lines. Embryos were collected from apple juice agar caps supplied with yeast paste for a 24-h window at 25°C. Embryos were carefully transferred to a mesh-bottom chamber using a fine paintbrush and then dechorionated with 50% bleach for 2 min, fully rinsed, and transferred to a 1.5-ml centrifuge tube containing 0.5 ml of heptane. Next, embryos were placed on a coverslip to make a single layer. Immediately after heptane evaporated, embryos were covered with a thin layer of halocarbon oil 27 (Sigma-Aldrich). The coverslip with embryos was then taped to a metal microscope slide with an open window in the middle. Images and videos were recorded as described above with Zeiss LSM800 upright microscope.
Halocarbon oil
Manufactured by Merck Group
Sourced in United States
Halocarbon oil is a specialty fluid designed for use in laboratory equipment. It is a clear, colorless liquid with a low viscosity and high thermal stability. Halocarbon oil is inert and non-reactive, making it suitable for use in a variety of laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Axio vert a1 inverted microscope, by Narishige (2 mentions)
Micromanipulator mmo 4, by Narishige (2 mentions)
Moflo astrios eq, by Beckman Coulter (2 mentions)
Calcein violet 450 am, by Thermo Fisher Scientific (2 mentions)
7 aad, by Thermo Fisher Scientific (2 mentions)
Lsm 800 upright microscope, by Zeiss (1 mentions)
Dmlb microscope, by Leica (1 mentions)
Zyla 4.2 plus scmos, by Oxford Instruments (1 mentions)
Lumox dish, by Sarstedt (1 mentions)
Emccd camera, by Oxford Instruments (1 mentions)
Dragonfly 200, by Oxford Instruments (1 mentions)
Micro knife, by Fine Science Tools (1 mentions)
Lightsheet z 1, by Zeiss (1 mentions)
Zen 2014 sp software, by Zeiss (1 mentions)
Aqua poly mount, by Polysciences (1 mentions)
Low melting agarose, by Merck Group (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
18 protocols using halocarbon oil
1
Microtubule Polarity Dynamics in Developing Neurons
2
Detailed Characterization of Ecdysis Behavior
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Detailed characterization of ecdysis behavior was done using puparium-free preparations, as described in Kim et al. (2006) (link). Briefly, immediately after the first signs of onset of ecdysial behaviors the pupa was surgically removed from the puparium in a drop of PBS (137 mM NaCl, 2,7 mM KCl, 10 mM Na2HPO4 and 2 mM KH2PO4, pH 7.3), and placed in a recording chamber filled with halocarbon oil (Sigma-Aldrich Chemical Co., MO) in order to prevent desiccation. The animals were filmed at room temperature (20–22°C) under dim transmitted light using a Leica DMLB microscope (20 X magnification).
3
Actin Depolymerization in C. elegans
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The Lat-A injections for actin depolymerization were performed as described previously (Chia et al., 2012 (link)). L4 stage animals were immobilized by putting them on 2% agarose pads and adding halocarbon oil (Sigma). After immobilization, the C. elegans were injected with either 1mM of LAT-A (Sigma) in 25% vol/vol DMSO (Sigma) or 25% DMSO alone. The injections were done into the pseudocoelom of the C. elegans at a site slightly posterior to the vulva. The injected animals were then kept at 20°C for 3h to recover. After recovery, the animals were imaged as has been described above.
4
Actin Depolymerization in C. elegans
5
Live Imaging of Drosophila Embryos
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Embryos were laid on apple juice agar plates for approximately 1 h and embryos were collected and dechorionated in 50% bleach solution (2.5% final concentration of sodium hypochlorite solution diluted in water). Preparation of embryos for live imaging was performed as described,77 (link) with embryos mounted onto a heptane glue coated coverslip (Scientific Laboratory Supplies, Cat# MIC3110) and inverted over a coverslip bridge in a 7:1 ratio mix of 700:27 halocarbon oil (Sigma, Cat# H8773 and Cat# H8898) on the membrane of a Lumox dish (Sarstedt AG & Co, Cat# 94.6077.305). Images were collected on an Andor Dragonfly200 spinning disk upright confocal microscope with a 40x/1.30 HCL pL Apochromat objective. Samples were excited using 488nm (11%; sogMS2, gtMS2 and mewMS2 or 13%; hntMS2) and 561nm (6%) diode lasers via Leica GFP and RFP filters respectively. Images were collected simultaneously using dual camera imaging with Zyla 4.2 Plus sCMOS (2048 X 2048) and iXon EMCCD camera (1024 X 1024) with a gain of 180 and binning [2X and 1X respectively] for 130ms. For each movie a total of 50 Z stacks at 0.7μm spacing were collected using the fastest setting yielding a total Z size of 35 μm at a time resolution of between 20 and 25 s on average.
6
Generating Transgenic C. elegans Strains
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To generate transgenic lines for localization, rescue experiments, and expression patterns, we generated the following transgenic animals via microinjections.
OIK1042 turEx21[arl-13p::wdr-31 (T05A8.5)::GFP::unc-54 3′UTR +rol-6}; T05A8.5(syb1568)II., elmd-1(syb630) II, rpi-2(K08D12.2)(ok1863) IV. (1 ng).
OIK1044 N2;turEx23[elmd-1p::GFP::elmd-1 (C56G7.3)::unc-54 3′UTR +rol-6} (5 ng).
OIK1045 N2;turEx24[arl-13p::GFP::elmd-1 (C56G7.3)::unc-54 3′UTR +rol-6} (5 ng).
OIK1046 N2;turEx25[elmd-1p(C56G7.3)::GFP::unc-54 3′UTR +rol-6} (50 ng).
OIK1047 N2;turEx26[wdr-31 (T05A8.5)p::GFP::unc-54 3′UTR +rol-6} (50 ng).
The rol-6 plasmid (50 ng/μl plasmid pRF4) was co-injected as the co-transformation marker. In brief, the plasmids were delivered by microinjections into the gonads of 1-d adult worms. Worms were initially transferred onto a 2.5% agarose pad before (Halocarbon oil, 9002-83-9; Sigma-Aldrich), followed by microinjection. The microinjection was done using a Zeiss Axio Vert.A1 inverted microscope with DIC optics coupled with a Narishige Micromanipulator MMO-4. We next manually inspected the plates to find successful transgenic animals.
OIK1042 turEx21[arl-13p::wdr-31 (T05A8.5)::GFP::unc-54 3′UTR +rol-6}; T05A8.5(syb1568)II., elmd-1(syb630) II, rpi-2(K08D12.2)(ok1863) IV. (1 ng).
OIK1044 N2;turEx23[elmd-1p::GFP::elmd-1 (C56G7.3)::unc-54 3′UTR +rol-6} (5 ng).
OIK1045 N2;turEx24[arl-13p::GFP::elmd-1 (C56G7.3)::unc-54 3′UTR +rol-6} (5 ng).
OIK1046 N2;turEx25[elmd-1p(C56G7.3)::GFP::unc-54 3′UTR +rol-6} (50 ng).
OIK1047 N2;turEx26[wdr-31 (T05A8.5)p::GFP::unc-54 3′UTR +rol-6} (50 ng).
The rol-6 plasmid (50 ng/μl plasmid pRF4) was co-injected as the co-transformation marker. In brief, the plasmids were delivered by microinjections into the gonads of 1-d adult worms. Worms were initially transferred onto a 2.5% agarose pad before (Halocarbon oil, 9002-83-9; Sigma-Aldrich), followed by microinjection. The microinjection was done using a Zeiss Axio Vert.A1 inverted microscope with DIC optics coupled with a Narishige Micromanipulator MMO-4. We next manually inspected the plates to find successful transgenic animals.
7
Pupal Wing Imaging and Time-lapse Analysis
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Prepupae of indicated genotypes were raised and collected at room temperature, then shifted to 29 °C until staged appropriately (late inflation stage, roughly 18 h AP equivalent at 25 °C). Pupae were retrieved, briefly rinsed in water, dried on a Kimwipe, then positioned on a piece of double-sided tape (right wing facing up). Windows were carefully dissected into the pupal cases in the region of the wing using a microknife (cat# 10316–14; Fine Science Tools) essentially as described (51 (link)), avoiding damage to the underlying tissue. A tiny drop of halocarbon oil (Sigma Aldrich) was applied to the exposed pupal wing with a disposable pipet tip to prevent tissue desiccation during imaging. The pupae, adhering to strips of double-sided tape cut with a disposable scalpel, were then placed oil-side down onto a 24 × 50 mm coverslip. After 5–6 pupae were collected onto the coverslip, wings were time-lapse imaged on a Leica SP8 STED confocal microscope by taking optical anteroposterior cross sections of each wing every 4–5 min using the xzyt-function. The resulting time lapse images were processed into AVI-format videos using Imaris v.9.1.2 (Bitplane/Oxford Instruments).
8
Lightsheet Imaging of Zebrafish Embryos
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Embryos at stage 11 were selected using halocarbon oil (Sigma), dechorionated and mounted into the Lightsheet Z.1 (Carl Zeiss, Oberkochen, Germany) microscope, and imaged with a 40× W Plan-Apochromat 40× 1.0 UV–VIS detection objective [30 (link),50 (link),51 (link)]. Image data were processed using the maximum intensity projection function of ZEN 2014 SP software (Carl Zeiss, Oberkochen, Germany).
9
Analyzing Drosophila Embryo Hatching
10
Imaging Embryo Constriction Dynamics
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For fixed embryo imaging, single-plane images were collected on a confocal microscope (LSM710; ZEISS) using a 40× water-immersion objective, NA 1.2, with the pinhole set to one airy unit, and resolution of 1,024 × 1,024 pixels. For live-embryo imaging, the microscope and settings were the same except imaging was done on a 25°C heated stage. Mounting media was Aqua-Poly/Mount (Polysciences) for fixed embryos and halocarbon oil (Sigma-Aldrich) for live embryos. To follow constriction rates, time-lapse images were collected en face (i.e., in surface views) at 2.5-min intervals. This interval allowed refocusing of the imaging plane as the rings moved progressively deeper into the embryo. To follow FRAP, time-lapse images were collected in cross section at 1-s intervals. A 1-µm × 1.5-µm box, encompassing a ring in profile, was photobleached with a 561-nm laser at 100% power for 2 s, to reach ∼50% of the original fluorescence intensity. Recovery of the bleached ring was monitored for the next 150 s, until the fluorescence intensity stabilized at a plateau. Three unbleached rings were simultaneously monitored.
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