Imaging experiments were conducted and analyzed as previously described [30 (link)]. Briefly, freshly prepared adrenal sections from heterozygous Cyp11b2(AS)Cre/+ male mice expressing either a zG-targeted Ca2+ indicator (GCaMP3) or archaerhodopsin-based voltage indicator (Optopatch) were perfused in a PIPES buffer (in mM: 20 PIPES, 122 NaCl, 3 KCl, 1.25 CaCl2, 1 MgCl, 25 D-Glucose, 0.1% BSA, pH 7.3) containing Ang II: 3nM (GCaMP3) or 1μM (Optopatch), and imaged using a Zeiss Axio-Examiner spinning disk confocal (GCaMP3, 488nm laser) or widefield (Optopatch, 637nm laser) microscope (Intelligent Imaging Innovations, Inc. [3i]). Images were acquired at 63x using Slidebook 6 (3i) software at 40Hz (GCaMP3, ~10 minutes) or 100Hz (Optopatch, ~2 minutes) with a sCMOS camera (Hamamatsu Orca-Flash 4.0). Image series were exported as multipage TIFF files, and fluorescent intensity/time extracted from regions of interested delineating in-focus zG cells using Caltracer software (Yuste lab, Columbia U., Matlab).
Axio examiner
Manufactured by Zeiss
Sourced in Germany, United Kingdom
The Axio Examiner is a high-performance microscope system designed for advanced imaging and analysis. It features a modular design with a range of interchangeable components, allowing for customization to meet specific research requirements. The Axio Examiner provides superior optical quality, enabling detailed observation and precise imaging of a variety of samples.
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Lab products found in correlation
Lsm 780, by Zeiss (4 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (3 mentions)
Fluo 4 am, by Thermo Fisher Scientific (3 mentions)
Oregon green 488 bapta 1, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Multiclamp 700b, by Molecular Devices (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Pluronic f 127, by Thermo Fisher Scientific (2 mentions)
Orca flash 4, by Hamamatsu Photonics (1 mentions)
Plan apochromat 20 0.8 air objective, by Zeiss (1 mentions)
Axio imager z1, by Zeiss (1 mentions)
Airyscan, by Zeiss (1 mentions)
P4762, by Merck Group (1 mentions)
Sylgard coated, by Dow (1 mentions)
Pclamp software, by Molecular Devices (1 mentions)
Axiocam mr3, by Zeiss (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Tunel apoptosis detection kit, by Vazyme (1 mentions)
Flash 4 camera, by Hamamatsu Photonics (1 mentions)
34 protocols using axio examiner
1
Imaging Adrenal zG Cells in Mice
2
Fluorescence Microscopy for Polymersome Imaging
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Upon polymersome formation, the sample was imaged by fluorescence microscopy using a ZEISS Axio Examiner, fixed stage upright microscope, equipped with a 20× (0.5 numerical aperture) dipping objective which aided in visualization of the vesicles located at the bottom of the rehydration well, as depicted in Figure 1 B. During the irradiation step, the fluorescent cube was rotated out of position to allow the beam to pass through to the objective unobstructed, and then moved back into position to allow for post-irradiation monitoring of the vesicle and its fluorescent encapsulants.
3
Whole-mount Immunohistochemistry of Zebrafish
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For whole-mount immunohistochemistry, zebrafish embryos were deyolked and mounted onto glass slides with 70% glycerol. For synapse quantifications, images were acquired using a Zeiss LSM880 with Airyscan, Axio examiner confocal microscope with Plan-Apochromat 20×/0.8 air objective.
To image abnormal motor axons in response to various combinations of drug treatment, a Zeiss AxioImager Z1, with ApoTome.2 upright microscope was used.
To image abnormal motor axons in response to various combinations of drug treatment, a Zeiss AxioImager Z1, with ApoTome.2 upright microscope was used.
4
Electrophysiological Recordings in Neuronal Cells
5
Intracellular Calcium Imaging in Myotubes
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Intracellular Ca2+ levels were monitored in myotubes using the Fluo4-acetoxymethyl ester dye (Fluo4/AM; Life Technologies). An upright microscope (Zeiss Axio Examiner) was used, equipped with a 40 × 0.75NA water-immersion objectives and connected by an optical fiber to a 75W Xenon lamp and a monochromator (OptoScan; Cairn Instrument, UK). The fluo4-loaded cells in normal external solution (140 mM NaCl, 2.8 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM glucose, 10 mM Hepes, pH 7.3) were excited at 488 nm, and the fluorescence images were acquired at 5 frames/s with a 16 bit digital EMCCD camera (PhotoEvolve 512; Photometrics; Tucson, AZ, USA).
The temporal analysis was calculated as the mean fluorescence intensity signal in a selected cell area, as f/f0, where f is the fluorescence emission of a single loaded cell that was acquired during the time lapse, and f0 is the mean fluorescence intensity of the same cell calculated from images acquired during the first 5 s. The time to peak was also recorded, and the velocity to the peak was calculated as the ratio between the peak amplitude and the time to peak.
The temporal analysis was calculated as the mean fluorescence intensity signal in a selected cell area, as f/f0, where f is the fluorescence emission of a single loaded cell that was acquired during the time lapse, and f0 is the mean fluorescence intensity of the same cell calculated from images acquired during the first 5 s. The time to peak was also recorded, and the velocity to the peak was calculated as the ratio between the peak amplitude and the time to peak.
6
Intact Olfactory Network Brain Preparation
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The brain preparation leaving the entire olfactory network intact has been described previously (Demmer and Kloppenburg, 2009 (link); Husch et al., 2009a (link); Kloppenburg et al., 1999 (link)). Animals were anesthetized by CO2, placed in a custom-built holder, and the head was immobilized with tape (tesa ExtraPower Gewebeband, Tesa, Hamburg, Germany). The head capsule was opened by cutting a window between the two compound eyes and the antennae’s bases. The brain with its antennal nerves and attached antennae was dissected in extracellular saline (see below) and pinned in a Sylgard-coated (Dow Corning Corp., Midland, MI) recording chamber. To get access to the recording site, we desheathed parts of the AL using fine forceps, and preparations were enzymatically treated with a combination of papain (0.3 mg·ml–1, P4762, Sigma) and L-cysteine (1 mg·ml–1, 30090, Fluka) dissolved in extracellular saline (~3 min, room temperature [RT], ~24°C). For electrophysiological recordings, the somata of the AL neurons were visualized with a fixed stage upright microscope (AxioExaminer, Carl Zeiss, Jena, Germany) using a 20× water-immersion objective (20× W Apochromat, NA = 1) with a 4× magnification changer, and infrared differential interference contrast optics (Dodt and Zieglgänsberger, 1994 (link)).
7
Imaging Blebbing in U251 Cells
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U251 cells were washed twice with PBS and treated with 3 μM of AgNO3 in Ringer’s solution. Pictures of cells were taken every 30 seconds at 20× magnification using an Axio Examiner (Zeiss) with CCD Axiocam 502 mono digital camera (Zeiss) for at least 30 minutes. Blebs number and area were measured using Zen 2 image processing software.
8
Patch-Clamp Analysis of Inhibitory and Excitatory Synaptic Currents
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Recording pipettes (3.2–4.5 MΩ) were guided to iPS cells identified by GFP in the striatum or overlying cortex. Neurons were visualized using Dodt gradient contrast (x40 water immersion lens) and filter set 38 on an Axio Examiner fixed stage microscope (Zeiss, Thornwood, NJ) with digital camera (Rolera EM‐C2, Q imaging, Surrey, BC). Pipettes were filled with a low Cl‐ intracellular solution containing (mM): 6 NaCl, 4 NaOH, 130 K‐gluconate, 11 EGTA, 1 CaCl2, 1 MgCl2, 10 HEPES, 2 Na2ATP, and 0.2 Na2GTP Na2GTP and 0.5% biocytin (pH 7.3 and 296 mOsm). As a consequence, ECl = −69mV, inhibitory postsynaptic currents (IPSCs) had small amplitudes at VH = −60mV, though more prominent outward current amplitudes were achieved by shifting to VH = −40mV in some cases. All recordings were made in open, whole cell patch configuration under voltage clamp using a Multiclamp 700B (Molecular Devices, Sunnyvale, CA). Signals were sampled at 20 kHz and filtered at 10 kHz using p‐Clamp software (version 10.3, Molecular Devices, Sunnyvale, CA). After recordings, slices were fixed in 4% PFA and incubated for 2 hours with streptavidin‐555 (ThermoFisher) diluted 1:500 in PBS. Voltage clamp measurements. Cells were held at −60 mV and inward spontaneous excitatory postsynaptic currents (EPSCs) were recorded for 3 minutes. Cells were held at −40 mV and outward spontaneous IPSCs were recorded for 3 minutes.
9
Mitochondrial Membrane Potential Assay
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Cells were treated with different conditions: 6 h with 20 mM lactate or with 150 μM H2O2 for 30 min. After the initial treatments, 2.5 μM of JC-1 staining was added to the media for 10 min. Cells were washed in HBSS media (136 mM NaCl, 3 mM KCl, 1,25 mM CaCl2, 1,25 mM MgSO4, 10 mM HEPES and 2 mM D-glucose) and fluorescence levels were measured using a Zeiss LSM780 and AxioExaminer at λ = 530 nm and 590 nm.
10
Visualizing Retinal Waves via GCaMP6f
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Spontaneous Ca2+ transients during retinal waves are reflected by changes in the intensity of GCaMP6f signals expressed in the retinas of ChAT-Cre/Ai95D mice. GCaMP6f fluorescence signals were excited with 488 nm light under a conventional fluorescence microscope (Axio Examiner, Zeiss). Signals were then captured by an AxioCamMR3 camera and continuously visualized with AxioVision software (Zeiss). Real-time images were video recorded using the Open Broadcaster Studio software (bitrate: 2550 kbps, frame interval: 100 frames per second, output resolution: 1120 × 700 pixels) (free and open-source cross platform streaming and recording program: https://obsproject.com ). Images were acquired from OFF-SACs in the INL and ON-SACs in the GCL using a 63× or 20× water immersion objective lens. Although the INL is located deeply across the thickness of the retina, GCaMP6f signals of OFF-SACs were well visualized under the conventional fluorescence microscope. To limit crosstalk of GCaMP6f signals between ON and OFF SACs, we minimized intensity of excitation light when images were taken from each layer.
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