Gravid hermaphrodites were dissected in a watch glass filled with a 0.7 × dilution of Egg Salts medium (1 × medium is 118 mM NaCl, 40 mM KCl, 3.4 mM MgCl2, 3.4 mM CaCl2, 5 mM HEPES, pH 7.4). Embryos were mounted on a 2% agarose pad and covered with an 18 × 18 mm coverslip (No. 1.5H; Marienfeld). All imaging was performed in temperature-controlled rooms kept at 20°C. Two microscopes were used: a Zeiss Axio Observer microscope, equipped with an Orca Flash 4.0 camera (Hamamatsu) and a Colibri 2 light source (Zeiss), controlled by ZEN software (Zeiss); and a Nikon Eclipse Ti microscope coupled to an Andor Revolution XD spinning disk confocal system, composed of an iXon Ultra 897 CCD camera (Andor Technology), a solid-state laser combiner (ALC-UVP 350i; Andor Technology), and a CSU-X1 confocal scanner (Yokogawa Electric Corporation), controlled by Andor IQ3 software (Andor Technology).
Andor iq3
Manufactured by Oxford Instruments
Sourced in United Kingdom
The Andor IQ3 software is a comprehensive data acquisition and analysis platform for advanced imaging and spectroscopy applications. It provides a user-friendly interface for controlling a wide range of scientific cameras and spectrometers, enabling researchers to capture and analyze high-quality data efficiently.
Automatically generated - may contain errors
Lab products found in correlation
Csu x1 confocal scanner, by Yokogawa (4 mentions)
Orca flash 4.0 camera, by Hamamatsu Photonics (4 mentions)
Ixon ultra 897 ccd camera, by Oxford Instruments (3 mentions)
Zen software, by Zeiss (3 mentions)
Axio observer microscope, by Zeiss (3 mentions)
No 1.5h, by Marienfeld (2 mentions)
Dar 4m am, by Merck Group (2 mentions)
Dar 4m, by Cairn Research (2 mentions)
Eclipse ti, by Nikon (2 mentions)
Oil immersion objective, by Olympus (2 mentions)
Ixon3 emccd camera, by Oxford Instruments (2 mentions)
Ix81 inverted fluorescence microscope, by Olympus (2 mentions)
Ixon 897 emccd, by Olympus (2 mentions)
Uplansapo objective, by Olympus (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Eclipse ti microscope, by Nikon (1 mentions)
Ixonem du 897, by Oxford Instruments (1 mentions)
Ix81 inverted microscope, by Olympus (1 mentions)
Eclipse ti, by Oxford Instruments (1 mentions)
Orca er, by Hamamatsu Photonics (1 mentions)
18 protocols using andor iq3
1
Microscopic Imaging of Gravid C. elegans Embryos
2
Cilia Imaging and IFT Analysis in C. elegans
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All imaging was performed using young adult hermaphrodite worms, with the exception of the aging experiments, in which animals at larval stage 2 and 7/18 days after adulthood were also imaged for comparison. All animals used for imaging were immobilized using 5–10 mM levamisole and were placed on a 5% agarose pad mounted on a microscope slide.
Cilia imaging to generate signal-intensity distribution profiles was performed using an Axio Observer microscope (Zeiss) equipped with a Plan-Apochromat 63×/1.46 NA oil objective lens and an Orca Flash 4.0 camera (Hamamatsu) and controlled by Zen software (Zeiss). Z-stacks were acquired with 0.4 µm between each z-section.
Time-lapse imaging of IFT was performed using an Olympus IX81 inverted microscope coupled to an Andor Revolution XD spinning disk confocal system composed of an iXonEM+ DU-897 with 2× port coupler camera (Andor Technology), a solid-state laser combiner (ALC-UVP 350i; Andor Technology), and a CSU-X1 confocal scanner (Yokogawa Electric Corp.), controlled by Andor IQ3 software (Andor Technology). 200 frames were recorded for each phasmid cilium at 3 frames/s (333 ms per frame) using an UPLSAPO 100×/1.40 NA oil objective lens. All imaging was performed in temperature-controlled rooms kept at 20°C.
Cilia imaging to generate signal-intensity distribution profiles was performed using an Axio Observer microscope (Zeiss) equipped with a Plan-Apochromat 63×/1.46 NA oil objective lens and an Orca Flash 4.0 camera (Hamamatsu) and controlled by Zen software (Zeiss). Z-stacks were acquired with 0.4 µm between each z-section.
Time-lapse imaging of IFT was performed using an Olympus IX81 inverted microscope coupled to an Andor Revolution XD spinning disk confocal system composed of an iXonEM+ DU-897 with 2× port coupler camera (Andor Technology), a solid-state laser combiner (ALC-UVP 350i; Andor Technology), and a CSU-X1 confocal scanner (Yokogawa Electric Corp.), controlled by Andor IQ3 software (Andor Technology). 200 frames were recorded for each phasmid cilium at 3 frames/s (333 ms per frame) using an UPLSAPO 100×/1.40 NA oil objective lens. All imaging was performed in temperature-controlled rooms kept at 20°C.
3
Live Imaging of C. elegans Embryos
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Gravid hermaphrodite worms were dissected in a watch glass filled with Egg Salts medium (118mM KCl, 3.4 mM MgCl2, 3.4 mM CaCl2, 5 mM HEPES, pH 7.4), and embryos were mounted onto a fresh 2% agarose pad. Imaging was performed in rooms kept at 20°C. Embryos co-expressing GFP::histone H2B and GFP::γ-tubulin were imaged on an Axio Observer microscope (Zeiss) equipped with an Orca Flash 4.0 camera (Hamamatsu), a Colibri.2 light source, and controlled by ZEN software (Zeiss). Embryos expressing GFP::p50DNC-2, dynein heavy chainDHC-1::GFP, EBP-2::mKate2, and mCherry::RAB-5 were imaged on a Nikon Eclipse Ti microscope coupled to an Andor Revolution XD spinning disk confocal system composed of an iXon Ultra 897 CCD camera (Andor Technology), a solid-state laser combiner (ALC-UVP 350i, Andor Technology), and a CSU-X1 confocal scanner (Yokogawa Electric Corporation), controlled by Andor IQ3 software (Andor Technology).
4
NADH/NADPH Autofluorescence Imaging
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The autofluorescence of NADH and NADPH (which can be referred to as NAD(P)H) was imaged on a cooled CCD camera (Hamamatsu, Orca ER). The blue autofluorescence, emitted by the pyridine nucleotides NADH and NADPH in their reduced form, was excited using a 360-nm filter and emission was collected using a 455-nm filter. Confocal images were acquired using a Zeiss 510 UV laser scanning microscope system and a 40× objective. The application of 1 μM of the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) maximised the rate of respiration and oxidised the mitochondrial NADH pool in cells, resulting in a decrease of detected fluorescence (minimum = 0% for NADH). The subsequent application of 1 mM of the complex IV inhibitor sodium cyanide (NaCN) suppressed respiration, preventing NADH oxidation and allowing the NADH pool to be regenerated (maximum = 100% for NADH).17 (link) Quantitative analysis of the obtained images was performed cell by cell using the Andor IQ3 software (Andor Technology). The average was taken from n >3 independent experiments for each condition.
5
Quantifying Astrocytic Nitric Oxide
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Optical recordings of NO production in cultured astrocytes were performed using an inverted epifluorescence Olympus microscope, equipped with a cooled CCD camera (Clara model; Andor). The cells were loaded with the NO sensitive fluorescent probe DAR-4M-AM (Sigma; 10 µM, 30 min incubation at room temperature) or transduced to express the genetically encoded NO sensor geNOp. Recordings were performed in a custom-made flow-through imaging chamber at ~32°C in aCSF saturated with 95% O2 / 5% CO2 (pH 7.4). The rate of chamber perfusion with aCSF was 4 ml min-1. DAR-4M fluorescence was excited by using a Xenon arc lamp and an Optoscan Monochromator (Cairn Research) at 560/10 and the fluorescence emission was recorded at 590 nm. geNOp fluorescence was excited at 488/10 nm and the fluorescence emission was recorded at 535 nm.
Hypoxic conditions in vitro were induced by the displacement of oxygen in the medium by argon. In all experiments in cell cultures and organotypic slices the hypoxic challenge was applied for 5-15 min. A representative profile of PO2 changes in the recording chamber during argon displacement is illustrated byFigure 2F . All test drugs were applied ~10 min before the hypoxic challenge. Imaging data were collected and analyzed using Andor iQ3 software (Andor).
Hypoxic conditions in vitro were induced by the displacement of oxygen in the medium by argon. In all experiments in cell cultures and organotypic slices the hypoxic challenge was applied for 5-15 min. A representative profile of PO2 changes in the recording chamber during argon displacement is illustrated by
6
Imaging Centrosomes and Nuclei in C. elegans Embryos
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Adult gravid hermaphrodite worms were dissected in a watch glass filled with Egg Salts medium (118 mM KCl, 3.4 mM MgCl2, 3.4 mM CaCl2 and 5 mM HEPES pH 7.4), and embryos were mounted on a fresh 2% agarose pad and covered with an 18 mm×18 mm coverslip (No. 1.5H, Marienfeld). Embryos co-expressing GFP::histone H2B and GFP::γ-tubulin for tracking of centrosomes and nuclei (Fig. 1 F–H; Fig. 2 C–G; Fig. 3 G) were imaged on an Axio Observer microscope (Zeiss) equipped with an Orca Flash 4.0 camera (Hamamatsu), a Colibri.2 light source, and controlled by ZEN software (Zeiss). All other imaging was performed on a Nikon Eclipse Ti microscope coupled to an Andor Revolution XD spinning disk confocal system composed of an iXon Ultra 897 CCD camera (Andor Technology), a solid-state laser combiner (ALC-UVP 350i, Andor Technology), and a CSU-X1 confocal scanner (Yokogawa Electric Corporation), controlled by Andor IQ3 software (Andor Technology). All imaging was performed in temperature-controlled rooms kept at 20°C. Time-lapse sequences were processed and analyzed with Fiji software (Image J version 2.0.0-rc-56/1.51 h).
7
Live Imaging of Intracellular Parasites
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Tachyzoites were inoculated onto confluent HFF monolayers grown in 35-mm glass bottom dishes (23-mm glass Fluorodish; WPI) and incubated at 37°C and 5% CO2 in a humidified incubator for 16 to 24 h. Before imaging, the sample was transferred into a heated observation chamber (37°C) equilibrated with 5% CO2. Imaging was carried out with a 100× 1.4 numerical aperture oil immersion objective (Nikon) mounted on an inverted microscope (Eclipse-Ti; Nikon) equipped with a CSU-W1 spinning-disc confocal head (Yokogawa). Two solid-state lasers were used as the excitation sources at 488 nm (100 mW) and 561 nm (100 mW) for GFP and Tomato RFP, respectively. Fluorescence emission was filtered with a trio dichroic mirror (Andor Technology) at 525/30 nm and 607/36 nm for GFP and RFP, respectively. Images were acquired with a Neo sCMOS camera (Andor Technology); the acquisition process was controlled by Andor iQ3 software (Andor Technology). Images were collected every 10 min; postacquisition processing was performed in ImageJ.
8
Confocal Imaging of TRPV4-Mediated Ca2+ Signaling
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Time-lapse images were acquired using a confocal spinning disk (Yokogawa CSU-X, Tokyo, Japan) mounted on an inverted microscope equipped with an autofocus system (Ti-ECLIPSE; Nikon, Tokyo, Japan). Light sources used for fluorescence imaging included an argon ion laser (300 mW; Melles Griot), three diode-pumped solid-state lasers including 405 nm (100 mW; Andor, Belfast, Northern Ireland), 561 nm (50 mW; Andor), and 640 nm (100 mW; Andor). Images were captured with a high-sensitivity iXon Ultra EM-CCD camera (DU-897U; Andor) operated with the Andor iQ3 software (Andor). Cells were seeded on glass coverslips and recorded before and after TRPV4 agonist (20 nM GSK1016790A or 5 μM 4α-PDD) application. The confocal images of labeled cells were collected using a 60× oil objective. Nuclear was stained with hoechst 33342 (H1399, Invitrogen) for 10 min. Di-8 ANEPPS (D3167, Invitrogen) was applied for 20 min as a marker for plasma membrane. Fluo-4, AM was used to measure intracellular Ca2+, for which cells were incubated in the dye solution at room temperature for 1 h.
9
Imaging Dividing Spermatocytes in C. elegans
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Embryos co-expressing mCherry::HIS-11 and GFP::TBB-2 or expressing GFP::DHC-1 were 519 isolated and mounted as described above. Imaging was performed at 20 ºC on a Nikon Eclipse 520 Ti microscope coupled to an Andor Revolution XD spinning disk confocal system, composed 521 of an iXon Ultra 897 CCD camera (Andor Technology), a solid-state laser combiner (ALC-UVP 522 350i, Andor Technology), and a CSU-X1 confocal scanner (Yokogawa Electric Corporation). 523
The system was controlled by Andor IQ3 software (Andor Technology). Images were acquired 524 with a 60x NA 1.4 Plan-Apochromat objective at 1 x 1 binning. 525 526 Fluorescence imaging of dividing spermatocytes 527 L4 hermaphrodites or young adult males were paralyzed with in a 5-µL drop of 5 mM 528 levamisole in M9 buffer for 10 min on an 18 mm x 18 mm coverslip and mounted on a fresh 529 5 % agarose pad. Imaging was performed on the spinning disk confocal system described 530 above with a 60x NA 1.4 Plan-Apochromat objective at 1 x 1 binning. 531 532
The system was controlled by Andor IQ3 software (Andor Technology). Images were acquired 524 with a 60x NA 1.4 Plan-Apochromat objective at 1 x 1 binning. 525 526 Fluorescence imaging of dividing spermatocytes 527 L4 hermaphrodites or young adult males were paralyzed with in a 5-µL drop of 5 mM 528 levamisole in M9 buffer for 10 min on an 18 mm x 18 mm coverslip and mounted on a fresh 529 5 % agarose pad. Imaging was performed on the spinning disk confocal system described 530 above with a 60x NA 1.4 Plan-Apochromat objective at 1 x 1 binning. 531 532
10
Measuring Astrocytic Nitric Oxide Dynamics
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Optical recordings of changes in NO production in cultured astrocytes were performed using an inverted epifluorescence Olympus microscope, equipped with a cooled CCD camera (Clara, Andor, Oxford Instruments). The cells were loaded with NO sensitive fluorescent probe DAR-4M-AM (Sigma; 10 µM for 30 min incubation at room temperature) or transduced to express genetically encoded NO sensor geNOp (NGFI; Austria). After incubation with the dye the cultures were washed three times with aCSF prior to the experiment. Recordings were performed in a custom-made flow-through imaging chamber at ~32°C in aCSF saturated with 95% O2 / 5% CO2 (pH 7.4). The rate of chamber perfusion with aCSF was 4 ml min -1 . To record changes in cytosolic [NO], DAR-4M or geNOp fluorescence were excited by using a Xenon arch lamp and a Optoscan Monocromator (Cairn Research) at 560/10 and 488/10 nm and the florescence emission was recorded at 590 and 535 nm, respectively. Hypoxic conditions in vitro were induced by displacement of oxygen in the medium with argon.
All test drugs were applied ~10 min before the hypoxic challenge. Imaging data were collected and analyzed using Andor iQ3 software (Andor, Belfast, UK). All data presented were obtained from at least 6 separate experiments.
All test drugs were applied ~10 min before the hypoxic challenge. Imaging data were collected and analyzed using Andor iQ3 software (Andor, Belfast, UK). All data presented were obtained from at least 6 separate experiments.
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