Methods are described previously (Briggman and Euler, 2011 (link)). Briefly, retinas were electroporated with OGB-1 (Invitrogen) using ten 13–14 V, 10-ms-pulse-width, 1-Hz-pulse-frequencysquarewave pulses. The procedure was performed under dim red illumination. Two-photon fluorescence images were obtained with a modified movable objective microscope (MOM) (Sutter Instruments) equipped with through-the-objective UV light stimulation (single LED NC4U134A, peak wavelength 385 nm; Nichia) with the laser tuned to 800 nm. Two kinds of light stimuli were presented while imaging: a series of flashed spots (231 m diameter) to categorize DSGCs as either On or On-Off and a bar (200 * 350 μm) moving in eight different directions across the field of view at 0.5 mm/s. Each direction was repeated three times.
Ogb 1
Manufactured by Thermo Fisher Scientific
Sourced in United States
OGB-1 is a calcium-sensitive fluorescent dye used for measuring intracellular calcium levels in cells. It is a green-fluorescent indicator that exhibits an increase in fluorescence upon binding to calcium ions.
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Lab products found in correlation
Alexa 594, by Thermo Fisher Scientific (4 mentions)
Axoclamp 2b amplifier, by Molecular Devices (4 mentions)
Pluronic f 127, by Thermo Fisher Scientific (3 mentions)
Ogb 5n, by Thermo Fisher Scientific (2 mentions)
Oregon green bapta 1 am ogb 1, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 549, by Thermo Fisher Scientific (1 mentions)
Oregon green bapta 1, by Thermo Fisher Scientific (1 mentions)
Calcium calibration buffer kit 1, by Thermo Fisher Scientific (1 mentions)
Alexa 568, by Merck Group (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Maitai deepsee, by Spectra-Physics (1 mentions)
Pluronic acid, by Thermo Fisher Scientific (1 mentions)
Magnesium green, by Thermo Fisher Scientific (1 mentions)
ω agatoxin iva, by Alomone (1 mentions)
ω conotoxin gvia, by Alomone (1 mentions)
Igor pro, by Wavemetrics (1 mentions)
Borosilicate glass capillaries, by World Precision Instruments (1 mentions)
Pclamp, by Molecular Devices (1 mentions)
P80 pipette puller, by Sutter Instruments (1 mentions)
Digidata 1550b, by Molecular Devices (1 mentions)
21 protocols using ogb 1
1
Two-Photon Imaging of Retinal Ganglion Cells
2
In Vivo Calcium Imaging of Cortical Neurons and Astrocytes
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The mice were anesthetized and surgically operated by methods similar to those in electrophysiology section. The dura was intact except for a few tiny holes made by glass pipette for dye injections. The injuries to the cerebral cortices and surface vessels were avoided (Zhao et al., 2012 (link)). Ca2+ dye, Oregon Green BAPTA-1-AM (OGB-1, Invitrogen USA), was applied to monitor the activities of the cortical neurons and astrocytes. OGB-1 was dissolved in DMSO and 20% Pluronic F-127 (Invitrogen, USA) for stock solution at 10 mM. This stock solution was diluted in the ACSF to yield final concentration at 1 mM, which was injected into layer I–II of the barrel cortices by the pressure (1 bar, 5 min) through glass pipettes (100 μm below the pia) to label the multiple cells. In the meantime, 100 μM sulforhodanmine-101 (SR101, Invitrogen) was co-injected to label the astrocytes (Zhao et al., 2012 (link)). The volumes of the dyes were controlled at −0.5 μl. After the injections, a craniotomy well was filled by low-melted agarose (1%) in the ACSF and sealed with a glass cover-slip. The exposed skull was adhered to a custom-made metal recording chamber with dental acrylic cement and superfused with the ACSF (in mM): 125 NaCl, 2.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2 and 20 glucose (pH 7.4) at 37°C and bubbled with 95%O2/5% CO2 (Zhang et al., 2012 (link)).
3
PVN Neuron Ca2+ Imaging Dynamics
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Parvocellular neurons of the PVN (N = 9) were patched as described above and individually loaded with OGB-1 (20 μM; Invitrogen) Ca2+-indicator fluorescent dye via whole-cell patch clamp configuration. The experimental conditions in the case of each recorded cells are as follows: firing protocol to test the viability of neurons, NPY treatment (1 μM) in the presence of TTX (600 nM) followed by glutamate (100 μM) treatment to test again the viability of neurons.
To test the mechanisms of the NPY induced increase of the intracellular Ca2+ levels, after the firing protocol, the sections were pretreated with TTX combined either with a PLCβ inhibitor, U73122 (5 μM), or a specific ryanodine receptor inhibitor, Dantrolene (5 μM). These pretreatments lasted at least 15–20 min before the recording started. Every treatment period was preceded with a control period of recording. Both Ca2+-imaging and electrophysiological data were analyzed offline. Neurons that did not show increase of OGB-1 fluorescence intensity during firing or after glutamate treatment were excluded from the analyses. The experimental procedure is described in details in the Supplementary Methods.
To test the mechanisms of the NPY induced increase of the intracellular Ca2+ levels, after the firing protocol, the sections were pretreated with TTX combined either with a PLCβ inhibitor, U73122 (5 μM), or a specific ryanodine receptor inhibitor, Dantrolene (5 μM). These pretreatments lasted at least 15–20 min before the recording started. Every treatment period was preceded with a control period of recording. Both Ca2+-imaging and electrophysiological data were analyzed offline. Neurons that did not show increase of OGB-1 fluorescence intensity during firing or after glutamate treatment were excluded from the analyses. The experimental procedure is described in details in the Supplementary Methods.
4
Two-Photon Imaging of VPC Calcium Dynamics
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Fluorescence was recorded by two-photon laser scanning microscopy on a Femto-2D microscope (Femtonics, Budapest, Hungary), equipped with a tunable, Verdi-pumped Ti:Sa laser (Chameleon Ultra I, Coherent, Glasgow, Scotland). The microscope was equipped with a 60x Nikon Fluor water-immersion objective (NA 1.0; Nikon Instruments, Melville, NY, USA), three detection channels (green fluorescence (epi and trans), red (epi) and infrared light (trans)), and controlled by MES v4.5.613 software (Femtonics).
VP-eGFP cells were identified in the green channel at an excitation wavelength of 950 nm. VPC bodies were patched in the whole-cell mode with patch pipettes filled with regular intracellular solution (see Electrophysiology), Alexa Fluor 549 (50 µM, Invitrogen) and the Ca2+ indicator OGB-1 (100 µM, Invitrogen, Thermo Fisher Scientific) were added for neurite visualization and Ca2+ imaging. Fluorescence transients and image stacks were acquired at 800 nm laser excitation. Data were mostly collected from the medial surface of the OB. Ca2+ imaging experiments were performed at room temperature (~21 °C). The patched VPCs were held in the current clamp mode near their resting potential of −55 mV. Structures of interest were imaged in free line-scanning mode with a temporal resolution of ~1 ms.
VP-eGFP cells were identified in the green channel at an excitation wavelength of 950 nm. VPC bodies were patched in the whole-cell mode with patch pipettes filled with regular intracellular solution (see Electrophysiology), Alexa Fluor 549 (50 µM, Invitrogen) and the Ca2+ indicator OGB-1 (100 µM, Invitrogen, Thermo Fisher Scientific) were added for neurite visualization and Ca2+ imaging. Fluorescence transients and image stacks were acquired at 800 nm laser excitation. Data were mostly collected from the medial surface of the OB. Ca2+ imaging experiments were performed at room temperature (~21 °C). The patched VPCs were held in the current clamp mode near their resting potential of −55 mV. Structures of interest were imaged in free line-scanning mode with a temporal resolution of ~1 ms.
5
Hippocampal Calcium Imaging with OGB-1
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Ca2+ activity was recorded with a confocal microscope, Zeiss LSM DuoScan 510, in CA1 str. radiatum of hippocampal slices pre‐incubated with Ca2+ dye, OGB‐1 (Invitrogen, USA) and an astrocyte‐specific marker, sulphorhodamine 101 (100 nM, Invitrogen, USA). After the preparation, the slices were transferred to a 3 ml incubation chamber with constantly gassed storage solution containing both dyes. OGB‐1 was initially dissolved to 0.795 mM in 0.8% Pluronic F‐127 in DMSO. Then, 3 µl of the dye was added to the chamber. After incubation for 40 – 45 min at 37°C in the dark, the slices were transferred to the recording/imaging chamber for time‐lapse imaging (one frame/s). OGB‐1 was excited with a 488 nm argon laser and imaged with an emission band‐pass filter 500 – 530 nm; sulphorhodamine 101 was excited with a 543 nm HeNe laser and imaged with an emission band‐pass filter 650 – 710 nm. The imaging was performed for 10 min at 34°C in normal ASCF, and then, 30 dark noise images were recorded.
6
Cytosolic Ca2+ Measurements in DA Neurons
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For cytosolic Ca2+ measurements in tissue slices, DA neurons were loaded with Alexa-594 (red dye, 30 µM, Invitrogen) and Oregon Green Bapta-1 (green dye, OGB-1, 200 µM, Invitrogen) together. Optical signals were acquired at 800 nm of two-photon excitation beam to simultaneously excite both dyes. ROI images were acquired during frame scanning (512 × 512 pixels) with 10–20 ms time intervals and Ca2+ levels from the ROIs were quantified as changes in green Ca2+ fluorescence from OGB-1 divided by morphological red fluorescence of Alexa-594 (G/R). Ratio between OGB-1 and Alexa-594 was used to minimize interference of the fluorescence photobleaching.
Acutely dissociated DA neurons were incubated with 3–5 µM Fluo 4-AM in high-glucose solution at room temperature (20°C–25°C) for 30 min. The fluorescence intensities of the neurons were measured using a Zeiss 510 confocal microscope (40× oil immersion objective lens or 60× water immersion objective lens). Fluo 4-AM Ca2+ indicators were excited at 488 nm (argon laser) and cytosolic Ca2+ signals were collected through 550 nm long-pass filter. Ca2+ level changes represented delta fluorescence intensity devided by basal level of fluorescence (ΔF/F0). To measure cytosolic Ca2+ concentration in some cases, we used a calibration kit (Calcium Calibration Buffer Kit #1; Invitrogen).
Acutely dissociated DA neurons were incubated with 3–5 µM Fluo 4-AM in high-glucose solution at room temperature (20°C–25°C) for 30 min. The fluorescence intensities of the neurons were measured using a Zeiss 510 confocal microscope (40× oil immersion objective lens or 60× water immersion objective lens). Fluo 4-AM Ca2+ indicators were excited at 488 nm (argon laser) and cytosolic Ca2+ signals were collected through 550 nm long-pass filter. Ca2+ level changes represented delta fluorescence intensity devided by basal level of fluorescence (ΔF/F0). To measure cytosolic Ca2+ concentration in some cases, we used a calibration kit (Calcium Calibration Buffer Kit #1; Invitrogen).
7
Calcium Imaging in Drosophila Larval Synapses
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Calcium imaging was performed as previously described (Müller and Davis, 2012 (link); Müller et al., 2015 (link)). Final stocks of OGB-1 488 (1 mM, Sigma) and Alexa-568 (5 mM, Sigma) were prepared in HL3 (0 mM Ca2+). Third instar Drosophila larvae was dissected and incubated on ice for 10 min in HL3 with zero calcium (1 mM OGB-1; 5 mM Alexa 488, Invitrogen). Indicators were removed and larvae were washed for 10 min with HL3, then placed into the recording chamber for imaging. A scanning confocal microscope (Ultima, Prairie Technologies) with a 60× objective (1.0 NA, Olympus) was used for imaging. 488 nm excitation wavelength from a krypton-argon laser used for excitation and emitted photons were collected through a pinhole at a photocathode photomultiplier tube (Hamamatsu). All line scans were performed at type 1b boutons of muscle 6/7, segments A2-A3. Loading efficiency of the dye was assessed by the intensity of co-loaded Alexa 568. Single stimuli (1 ms) and stimulus trains (5 pulse, 1 ms duration, 50 Hz) were used. Changes in the fluorescence were quantified as previously described (Müller and Davis, 2012 (link); Müller et al., 2015 (link)).
8
Calcium Imaging of Brain Slices
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For [Ca2+]i imaging 8–10 slices were incubated in a petri dish (5 ml) filled with of HBS (3.333 ml) containing Oregon Green 488 BAPTA-1 acetoxymethyl ester calcium fluorescent dye (6 μM, OGB-1, Invitrogen, Eugene, OR, USA), Pluronic F-127 (0.03% w/v) and dimethylsulphoxide (DMSO, 0.12% v/v) for 50 minutes on an orbital shaker (50 turns min−1) at room temperature and protected from light55 (link).
9
Ex Vivo Tumor Imaging via Two-Photon Microscopy
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A custom-modified two-photon microscope (Scientifica, Uckfield, UK) was used for the ex vivo LLC tumor imaging with a Ti:sapphire femtosecond laser (Mai Tai DeepSee, Spectra-Physics, US), galvo-resonant scanners, and a Nikon ×16/0.80 numerical aperture water-immersion objective. With 930-nm excitation wavelength, the emitted fluorescent signals from OGB-1 (Invitrogen, O6807) and tdTomato were detected by photomultiplier tubes through a 520/20-nm band-pass filter and 609/34-nm band-pass filter, respectively.
10
Preparation of Chemical Stock Solutions
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All chemicals were obtained from Sigma (Taufkirchen, Germany) or Carl Roth (Karlsruhe, Germany). OGB-1, Magnesium Green, and pluronic acid were obtained from Invitrogen (LifeTechnologies GmbH, Darmstadt, Germany). Tetrodotoxin (TTX), TTA-P2, ω-conotoxin GVIA, and ω-agatoxin IVa were obtained from Alomone Labs (Jerusalem, Israel). Stock solutions were prepared according to manufacturer’s instructions and stored at −20°C.
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