Two freshly laid egg clutches were kept for three days at 26 ºC incubator prior to preparatory procedures for time-lapse imaging. Eggs were dechorionated in 50% commercial bleach for 15 min, rinsed several times in PBS and glued to plastic petri dishes with double-sided tape. Embryos were then submerged in halocarbon 700 oil (Sigma-Aldrich) for imaging. Imaging was conducted at 20ºC–22 ºC. Images were taken every 15 min on a Nikon SMZ25. Videos of individual embryos have been cropped for clarity. For time-lapse 1 (Additional file 3 : File S3), five out of twelve embryos developed normally during the experiment. For time-lapse 2 (Additional file 4 : File S4), seven out of ten embryos developed normally during the experiment.
Halocarbon oil 700
Manufactured by Merck Group
Sourced in Germany
Halocarbon oil 700 is a high-performance lubricating fluid developed by Merck Group. It is a clear, colorless, and odorless liquid with a high boiling point and low vapor pressure. The product is designed to provide efficient lubrication and thermal stability in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Halocarbon oil 27, by Merck Group (6 mentions)
Lsm 510 meta confocal microscope, by Zeiss (4 mentions)
Uplans apo 1.3 na, by Olympus (3 mentions)
Glass bottom dish, by MatTek (2 mentions)
α amanitin, by Santa Cruz Biotechnology (2 mentions)
Mn 151, by Narishige (2 mentions)
Ultraview spinning disk system, by PerkinElmer (2 mentions)
Voltalef oil, by Avantor (2 mentions)
Femtotips 2 needles, by Eppendorf (2 mentions)
Orca fusion digital cmos camera, by Nikon (2 mentions)
Ix81 microscope, by Olympus (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Smz25, by Nikon (1 mentions)
Glass coverslip, by Thermo Fisher Scientific (1 mentions)
Schneider s drosophila medium, by Thermo Fisher Scientific (1 mentions)
Acridine orange, by Merck Group (1 mentions)
Puromycin, by Santa Cruz Biotechnology (1 mentions)
Csu 10, by Zeiss (1 mentions)
M205 fa, by Leica (1 mentions)
Dfc310 fx camera, by Leica (1 mentions)
63 protocols using halocarbon oil 700
1
Time-Lapse Imaging of Embryo Development
2
Imaging Meiotic Spindle Assembly in Drosophila
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Whole testes were dissected from 3rd instar larvae in Drosophila Schneider’s medium (Life Technologies) containing Antibiotic-Antomycotic (Life Technologies), and were mounted in the same medium for imaging on a 50 mm gas-permeable lumox dish (Sarstedt). The medium was surrounded by Halocarbon 700 oil (Sigma) to support a glass coverslip (22 × 22 mm, #1.5, Fisher) that was placed on top of the medium51 . For colchicine treatments, whole testes were placed in a glass-bottom dish (MatTek) filled with Schneider’s medium. Colchicine was then added to a final concentration of 50 μM at the indicated times. Individual cysts of spermatocytes in whole testes were imaged with an inverted Nikon A1R scanning confocal microscope system using 488 nm laser excitation for GFP and 568 nm laser excitation for RFP and tdTomato through a 40x, 1.3 NA objective lens. Z-stacks compromising 15–20 images at 1 µm intervals were obtained every 1 to 2 minutes. All imaging was carried out at room temperature. Images were processed and videos were compiled using ImageJ software (NIH). All image times are reported relative to NEB in order to standardize each timecourse to the same event for each cell. NEB was determined by the first image in the timecourse in which GFP-tubulin fluorescence appeared in the nucleus or the diffuse RFP-H2A fluorescence in the nucleus diminished.
3
Acridine Orange Staining of Apoptosis in D. sechellia Ovaries
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Apoptosis in D. sechellia ovaries (N > 6) was visualized following the protocol by Arama and Steller (McCall, 2004 (link)). Briefly, live ovaries were dissected in PBS and incubated in a freshly prepared solution of 0.6 μg/ml acridine orange (Sigma, St. Louis, MO) for 5 min, at RT. Ovaries were rinsed briefly in PBS and mounted in a drop of Halocarbon 700 oil (Sigma, St. Louis, MO) and observed immediately. Confocal images were obtained at 1-μm intervals over 20 μm Z-stack using a LSM510 Meta confocal microscope (Zeiss, Jena, Germany).
4
Live Imaging of Embryos with Pharmacological Manipulations
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Live imaging of embryos was conducted using a Yokogawa CSU10 spinning disc confocal microscope (Zeiss Observer.A1). Embryos were dechorionated in 4% bleach, washed with water, and mounted in halocarbon 27 Oil (Sigma). All live images were acquired in one minute intervals using Planapochrom 63X 1.4NA Oil objective. For pharmacological manipulations, embryos were lined up and glued to a coverslip, and desiccated for 12 to 13 minutes in a closed chamber containing drierite. Following desiccation, embryos were covered in halocarbon 700 oil (Sigma). Embryos were subsequently injected using a micromanipulator (Narishige, MN-151) with either water, 10,000 μg/mL puromycin (Santa Cruz Biotechnology), 1,000 μg/mL microbially sourced cycloheximide (Sigma), or 100 μg/mL α-amanitin (Santa Cruz Biotechnology) and imaged.
5
Time-lapse Imaging of Embryo Development
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Embryos were placed on oxygen-permeable film (lumox, Greiner Bio-one), affixed with dried heptane glue and then covered with Halocarbon 700 oil (Sigma) [43] . The lumox film was suspended on a copper plate that was temperature-regulated with two peltier plates controlled by an H-bridge temperature controller (McShane Inc., 5R7-570) with a thermistor feedback, accurate to 0.1°C. Time-lapse imaging with bright field transmitted light was performed on a Leica M205 FA dissecting microscope with a Leica DFC310 FX camera using the Leica Advanced Imaging Software (LAS AF) platform. Greyscale images were saved from pre-cellularization to hatch. Images were saved every one to five minutes, depending on the temperature. A humidifier was used to mitigate fluctuations in ambient humidity, though fluctuations did not affect developmental rate. Due to fluctuations in ambient temperature and humidity, the focal plane through the halocarbon oil varied significantly. Therefore, z-stacks were generated for each time-lapse and the most in-focus plane at each time was computationally determined for each image using an algorithm (implemented in Matlab) through image autocorrelation [44] (link), [45] . Time-lapse videos available from Dryad Digital Repository: doi:10.5061/dryad.s0p50.
6
Transcriptome Profiling of Drosophila Embryos
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Single His2Av-RFP(III), bratDf/fs1; His2Av-RFP(III) or brat heterozygous; His2Av-RFP(III) embryos (3–4 replicates per time point) were dechorionated in 100% bleach for 1′, mounted in halocarbon (700) oil (Sigma Aldrich, St. Louis, MO) on a coverslip, and imaged using a Nikon Ti-2e Epiflourescent microscope using a 60× objective. The nuclear cycle was identified following mitosis based on His2Av-RFP marked nuclear density (calculated by the number of nuclei/2,500 μm2). At the indicated time, embryos were picked into Trizol (Invitrogen, Cat #15596026) with 200 μg/ml glycogen (Invitrogen, Cat #10814010) and pierced with a 27G needle to release the RNA for 5 min. Late-stage single oocytes (4 replicates per genotype) were dissected from the ovaries of the mothers of the respective genotype and staged based on morphology. The oocytes were picked into Trizol (Invitrogen, Cat #15596026) with 200 μg/ml glycogen (Invitrogen, Cat #10814010) and pierced with a 27G needle to release the RNA for 5 min. RNA was extracted and RNA-seq libraries were prepared using the TruSeq RNA sample prep kit v2 (Illumina, San Diego, CA). Seventy-five base pair reads were obtained using an Illumina NextSeq500 sequencer at Northwestern Sequencing Core (NUSeq Core). This protocol is expanded in McDaniel and Harrison (2019) (link).
7
Thermal Stimulation of Drosophila Larvae
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Late third-instar larvae of OK6-Gal4/UAS-TRPA1 and control (OK6-Gal4/+) were raised at 22°C and transferred to a PCR tube containing 50 μl of Halocarbon 700 oil (Sigma-Aldrich). Intermittent stimuli consisting of a 3 min period at a higher temperature (27°C) separated by 5 min intervals at rest (22°C) were applied in the PCR machine for 8 h based on a published protocol (Pulver et al., 2009 (link)). The larvae were then transferred to regular medium for recovery before examination by immunostaining.
8
Imaging Live Drosophila Wing Discs
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Protocols were adapted from what was previously described in Handke et al. (2014) (link) and Poulton et al. (2014) (link). Briefly, wing discs were dissected from third instar larvae at room temperature and placed apical side up onto an oxygen-permeable Lumox dish 50 (Sarstedt) in a base media [Schneider’s media (GIBCO, Grand Island, NY), 5% fly extract (Drosophila Genomics Resource Center), 6.2 μg/ml bovine insulin, and 1% penicillin/streptomycin] supplemented with 200 μg/ml bovine insulin. Spacers of 1 μm in thickness were placed adjacent to the disc and a poly-l -lysine-coated coverslip was placed on top. Coverslips were sealed with Halocarbon oil 700 (Sigma Chemical, St. Louis, MO) around the edges. Discs were allowed to settle for 30 min, after which they were imaged with a 40× water objective for up to 3 hr.
9
Live Imaging of Drosophila Embryos
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All embryos were dechorionated in bleach (49 (link)) prior to being mounted ventral-side-up on double-sided tape (Scotch) in a minimal volume of Voltalef oil (VWR). All imaging of embryos was carried out on an UltraView Spinning Disk System (Perkin Elmer) using a 40x UplanSApo oil immersion objective lens (NA 1.3). The ventral region (most medial body segments) of embryos was imaged from the embryonic surface to a depth of 20µm, with z-slices spaced every 1µm. For lysotracker experiments requiring staining of live embryos, stage 15 dechorionated embryos were selected and transferred to a 50:50 mixture of peroxide-free heptane (VWR) and 10μM lysotracker red (Thermofisher) in PBS (Oxoid) in a glass vial, which was shaken in the dark for 30 minutes. Embryos were then transferred into a Watchmaker’s glass containing Halocarbon oil 700 (Sigma), before being mounted as described above.
Embryos requiring fixation and immunostaining were fixed and stained as previously described (17 (link)). For Fascin staining, embryos were treated with a mouse anti-Fascin primary antibody (sn7c; Developmental Studies Hybridoma Bank; used at 1:500), with Alexa fluor 568 goat anti-mouse used as a secondary antibody (A11031, Life Technologies; 1:200).
Embryos requiring fixation and immunostaining were fixed and stained as previously described (17 (link)). For Fascin staining, embryos were treated with a mouse anti-Fascin primary antibody (sn7c; Developmental Studies Hybridoma Bank; used at 1:500), with Alexa fluor 568 goat anti-mouse used as a secondary antibody (A11031, Life Technologies; 1:200).
10
Visualizing Drosophila Embryo Spindle Dynamics
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Imaging was performed on a Visitron Systems Olympus IX81 microscope with a CSO-X1 spinning disk using a UPlanS APO 1.3 NA (Olympus) 60× objective. Embryos 1–2 hr old were manually dechorionated, aligned in heptane glue on 22 × 50 mm coverslips, and covered with a 1:1 mixture of Halocarbon oil 700 and Halocarbon oil 27) (Sigma). Imaging was performed with 400 ms exposure per slice, with five slices per stack and a constant room temperature of 22°C. Embryos were injected using an Eppendorf Inject Man NI 2 and Femtotips II needles (Eppendorf). The anti-DSpd-2 and anti-Dgt6 antibodies were suspended in injection buffer (100 mM HEPES, pH 7.4, and 50 mM KCl), centrifuged at 13,500 × g for 20 min, and injected at a concentration of either 6 mg/ml (anti-Dgt6 or anti-DSpd-2, high concentration) or 1 mg/ml (anti-DSpd-2, low concentration). For cold-treatment assays, single embryos were imaged until metaphase was reached, at which point they were placed in 50 mm ice-cold Petri dishes and covered with 4°C Halocarbon oil. Following 90 min on ice, embryos were reimaged, with 30 s typically expiring between removal from 4°C and the initiation of imaging. In some cases, embryos were removed following 75 min on ice, injected with antibody, and then placed on ice for another 15 min prior to imaging.
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