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.
Lumox dish
Manufactured by Sarstedt
Sourced in Germany
The Lumox dish is a specialized tissue culture dish designed for high-performance cell culture applications. It features an optically clear, gas-permeable base that supports optimal cell growth and facilitates microscopic observation.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by Corning (2 mentions)
Sp8 inverted confocal microscope, by Leica (2 mentions)
Lsm710nlo two photon microscope, by Zeiss (2 mentions)
Glass bottom dish, by Ibidi (2 mentions)
Dragonfly 200, by Oxford Instruments (1 mentions)
Halocarbon oil, by Merck Group (1 mentions)
Zyla 4.2 plus scmos, by Oxford Instruments (1 mentions)
Emccd camera, by Oxford Instruments (1 mentions)
Lsm 5 pascal scanning confocal microscope, by Zeiss (1 mentions)
Dfc450 c camera, by Leica (1 mentions)
Mzfliii stereomicroscope, by Leica (1 mentions)
D5300 camera, by Nikon (1 mentions)
Inverted sp5 microscope, by Leica (1 mentions)
Csu 21, by Nikon (1 mentions)
Palmitic acid, by Merck Group (1 mentions)
Axiovert, by Zeiss (1 mentions)
Bioxtra, by Merck Group (1 mentions)
Dragonfly spinning disc confocal microscope, by Oxford Instruments (1 mentions)
7 protocols using lumox dish
1
Live Imaging of Drosophila Embryos
2
Live Imaging of Crk Knockdown Embryos
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Embryos with Crk knockdown and carrying live imaging constructs were produced by crossing either crkS-RNAi/CyO; His2av mRFP, sGMCA or crkS-RNAi/CyO; MRLC::GFP males to matα tub-GAL4 II; matα tub-GAL4 III females. crkS-RNAi/ matα tub-GAL4 II; His2av mRFP, sGMCA/matα tub GAL4 III or crkS-RNAi/ matα tub-GAL4 II; MRLC::GFP/matα tub GAL4 III female progeny were then crossed to crkS-RNAi males and the progeny were imaged. CyO/matα tub -GAL4 II; His2av mRFP, sGMCA/matα tub-GAL4 III females crossed to y, w males were used as controls. Flies were allowed to lay on apple juice agar plates with yeast for 2 h. Embryos were dechorionated in 50% bleach for 3 min, washed 3× with 0.01% Triton-X, and transferred to a fresh apple juice agar plate where they were immersed in halocarbon oil 27(Apex Bioresearch Products). Seven to 10 embryos were then transferred to halocarbon oil on the gas-permeable membrane of a Lumox dish (Sarstedt) and covered with a glass coverslip. Imaging was done on a Zeiss LSM-5 Pascal scanning confocal microscope (Oberkochen, Germany). Embryos were imaged at the apical cortex where the tops of actin caps were visible. Frames were taken at 10-s intervals for ∼2 h or until cellularization began.
3
Imaging of Quail Skin and Embryos
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Fixed flat skins and whole embryos were imaged using an AF-S Micro NIKKOR 60-mm f/2.8G ED macro-lens equipped with a D5300 camera (Nikon) and an MZ FLIII stereomicroscope (Leica) equipped with a DFC 450C camera (Leica). Confocal images were obtained using an inverted SP5 microscope (Leica) with 10× (dry) or 40× (immersed oil) objectives. For time-lapse imaging, dissected dorsal skin regions from membrane-GFP Japanese quails produced at the Pasteur Institute (Dr. Gros laboratory) at HH29, which corresponds to competence stage Co, were placed dermal side down on Millipore-nitrocellulose filters (Sigma #HABP04700) and cultured in a Lumox dish (⌀50 mm, Sarstedt #11008) containing 5 ml of DMEM supplemented with 2% FCS and 2% Penicillin/Streptomycin (and when applicable, 0.0625 μM/ml Latrunculin A) at 37°C in 5% CO2 atmosphere. Time-lapse images were acquired every 5 min up to 10 h with a SPINNING DISK-W1 (Zeiss) equipped with a 25× (immersed oil) objective.
4
Live Imaging of Germ Granules in Embryos
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Live imaging of founder granules and germ granules was performed on a Nikon Ti-E with Yokogawa spinning disc module (CSU-21) with a 60 × 1.4 NA oil immersion objective. Embryos were dechorionated and mounted in halocarbon oil 95 (Halocarbon Products Corporation) under a #1.5 glass coverslip (VWR) on a Lumox dish (Sarstedt), oriented with the dorsal side closest to the coverslip. Movies were acquired at 100 ms exposure in two channels with a frame rate of 5 frames per second. Particles were tracked using the Autoregressive Motion algorithm of Imaris version 9.1.2 (Bitplane).
5
Monitoring DAF-16::GFP Localization in Cadmium-Exposed Worms
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TJ356 worms were fed during growth with E. coli HT115 (TJ356, control RNAi and TJ356, pmk-1 RNAi). For an improved view on the cell nuclei of all tissues, worms without eggs were used for experiments (synchronized L3 larvae, which may have developed to L4 larvae during experiments). Plates with a thin NGM layer, seeded with E. coli HT115 and containing either 0 mmol/L (control condition) or 10 mmol/L (test condition) CdCl2, were kept overnight at 20 °C. Then, four ‘pads’ were cut out from the NGM layer using a drinking straw, placed on a gas permeable lumox® dish (Sarstedt, Nümbrecht, Germany), and surrounded with palmitic acid (10 mg/mL; BioXtra, Sigma-Aldrich, Germany) to prevent an escape of worms (Miller and Roth 2009 (link)). After lining the rim of the dish with a wet tissue to avoid desiccation and placing one worm on each pad, the dish was sealed with parafilm. Under a fluorescence microscope (Zeiss Axiovert), the TJ356 worms on the pads were regularly screened for DAF-16::GFP subcellular localization (cytoplasmic or intermediate and nuclear localizations) for a maximum of 8.5 h.
6
Imaging Skin Explant Dynamics
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Lateral skin explants were taken from E13.5 embryos (H2B-EGFPtg/wt) and transferred to media (advanced DMEM + 2 mM L-glutamine + 0.1 mg/ml penicillin/streptomycin + 10% FBS) at 37 °C where they formed rolls. The rolls were embedded in 1% low-melting agarose or Matrigel (Corning) in media at 37 °C, where the cutting edge was touching the membrane-bottom of a Lumox® dish (Sarstedt) or a glass-bottom dish (ibidi). The dishes were covered with media and incubated at 37 °C and 5% CO2 until imaging. Time-lapse imaging was carried out between E13.5 and E14.5 using an inverted SP8 confocal microscope (Leica microsystems), an inverted Dragonfly Spinning disc confocal microscope (Andor), or an inverted LSM710NLO Two-Photon microscope (Zeiss) with 20× air objective or 40× water or oil immersion objective and incubation at 37 °C and 5% CO2.
7
Imaging Embryonic Skin Explant Dynamics
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Lateral skin explants were taken from E13.5 embryos (H2B-EGFP tg/wt ) and transferred to media (advanced DMEM + 2 mM L-Glutamine + 0.1 mg/ml Penicillin/Streptomycin + 10% FBS) at 37 °C where they formed rolls. The rolls were embedded in 1 % low-melting agarose or Matrigel (Corning) in media at 37 °C, where the cutting edge was touching the membrane-bottom of a Lumox® dish (Sarstedt) or a glass-bottom dish (ibidi). The dishes were covered with media and incubated at 37 °C and 5 % CO2 until imaging. Time-lapse imaging was carried out between E13.5 and E14.5 using an inverted SP8 confocal microscope (Leica microsystems), an inverted Dragonfly Spinning disc confocal microscope (Andor) or an inverted LSM710NLO Two-Photon microscope (Zeiss) with 20x air objective or 40x water or oil immersion objective and incubation at 37 °C and 5 % CO2.
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