One week after surgery (postoperative day 5–7), rats were dark adapted for 30 min, anesthetized by CO2 and killed by cervical dislocation under dim red light (640 nm LEDs; Roithner Lasertechnik, Vienna, Austria). Isolated eyes were placed in a petri dish with oxygenated Ames' medium (A 1420, Sigma Aldrich, Germany) under a dissecting microscope illuminated with the dim red light. Eyes were hemisected, the vitreous body was carefully removed and the retina was peeled off the sclera. The retina was dissected into retinal portions, which extended well beyond the recording area of the electrode array. Retinal portions were mounted ganglion cell side down on the electrode array and were used for recordings immediately. The array surface was coated previously with poly-L-lysine hydrobromide (1 mg/ml in ultra-pure water, 150 kDa molecular weight; Sigma Aldrich, Germany). During recordings, retinae were constantly perfused with warm (35–37°C) oxygenated Ames' medium (A 1420, Sigma Aldrich, Germany) at a rate of 5–7 ml/min.
A1420
Manufactured by Merck Group
Sourced in Germany
The A1420 is a laboratory instrument designed for precise and efficient liquid handling. It features automated pipetting capabilities to assist in various laboratory workflows, such as sample preparation, reagent addition, and liquid transfers. The device operates based on standardized protocols to ensure consistent and reliable performance.
Automatically generated - may contain errors
Lab products found in correlation
C57bl 6j, by Jackson ImmunoResearch (2 mentions)
Tc 344b, by Warner Instruments (1 mentions)
Tc 324b, by Warner Instruments (1 mentions)
60mea200 30ir ito gr, by MultiSciences Biotech (1 mentions)
Millicell filter, by Merck Group (1 mentions)
Poly l lysine hydrobromide, by Merck Group (1 mentions)
Glass bottom dish, by MatTek (1 mentions)
Hawp01300, by Merck Group (1 mentions)
A0701, by Merck Group (1 mentions)
Vt1200, by Leica (1 mentions)
Matlab, by MathWorks (1 mentions)
12 protocols using a1420
1
Retinal Electrophysiology after Vitrectomy
2
Labeling and Imaging of Individual Retinal Ganglion Cells
3
Macaque Retina Maintenance and Imaging
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Eyes were removed from deeply anesthetized male and female macaque monkeys at the time of death (Macaca nemestrina, Macaca fascicularis, or Macaca mulatta) via the Tissue Distribution Program of the Washington National Primate Research Center and in accordance with protocols reviewed and approved by the University of Washington Institutional Animal Care and Use Committee. A total of 106 retinas acquired from 76 animals were used for this study. The retina was maintained in vitro by dissecting retina-choroid free of the vitreous and sclera in oxygenated Ames’ Medium (A1420; Sigma Chemical Co., St. Louis, MO) under light-adapted conditions78 (link),99 (link). The retina-choroid was placed flat, vitreal surface up, in a glass-bottomed superfusion chamber coated with poly-lysine mounted, choroid side down, on the microscope stage. The retina was continuously superfused with Ames’ medium (pH 7.3; oxygenated with 95% O2/5% CO2) and the temperature was thermostatically maintained within the chamber (TC-344B, Warner Instruments) at ~36 °C. The retina was observed under infrared illumination projected through the choroid from the microscope substage light source and visual stimuli were projected through the microscope optics through the objective lens.
4
High-density Retinal Electrophysiology
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The extracellular electrical activity of the retina was measured using a high-density CMOS microelectrode array comprising 128×128 equally spaced recording sensors which cover an area of 1 mm2. In this study, we measured every second column (128×64 sensors) with a sampling frequency of ∼10 kHz for each sensor. Details of the recording technique are described in [7] , [17] . During the recording, the retina was continuously superperfused with carbogenated Ames’ medium (A 1420, Sigma).
5
Retinal Electrophysiology in Mice
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The retinas of wild-type mice (C57BL/6J, Jackson Labs) were used for all experiments. The mice were dark adapted overnight and euthanized by cervical dislocation in accordance with all animal care standards provided by Northwestern University’s Institutional Animal Care and Use Committee. Lighting in animal facilities was kept on a 14/10 hour cycle, with lights on at 6:00 am. Typical retina in vitro times were 12:00 pm through 7:00 pm. For all experiments, mice of either sex (approximately 69% male), and ages P30-P90 were used; no differences in results were observed with sex or age. Eyes were dissected in oxygenated Ames medium at 32°C. Dissections were performed in complete darkness using infrared (IR, 900 nm) illumination and photo converters. In the experimental rig, retinas were mounted in a shallow dish, below a microscope objective and above a digital projector, in oxygenated Ames’ medium from Sigma-Aldrich (A1420) at 32°C at a flow rate of 10 ml/min. Two glass electrodes on headstage amplifiers were mounted on micromanipulators on either side. Cell-attached and current clamp experiments were performed as previously described26 (link).
6
Retinal Electrophysiology in Myopia Mouse Model
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C57BL/6 mice treated with 4-week form deprivation were sacrificed by spinal dislocation after dark-adapted overnight. Under dim red light, the eyes were enucleated immediately and put into Ames’ medium (A1420, Sigma-Aldrich), which was equilibrated with 95% O2 and 5% CO2. Retinas were dissected from the eyecups and mounted onto a piece of anodisc filter membrane (Anodisc 25, GE Health Bio-Sciences, USA) with photoreceptor side down. Mounted retinas were transferred into the recording chamber of a MEA chip (60MEA200/30iR-ITO-gr, Multi Channel Systems, Germany) with ganglion cell side toward the array and then continuously perfused with oxygenated Ames’ medium with the help of an ismatec peristaltic pump (78023-00, Cole-Parmer, USA) and maintained at 30° ± 2°C using a temperature controller (TC-324B, Warner Instruments, USA).
7
Wholemount Retina Preparation for Physiological Experiments
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Methods for wholemount tissue preparation have been described in detail previously58 (link). After the animal was euthanized, its eyes were enucleated and placed in bicarbonate-buffered Ames solution (Ames; Sigma, A1420), equilibrated to pH 7.4. It has been shown that variations in the O2 level in brain tissue can affect functional hyperemia5 (link). To reduce any discrepancy, O2 level was maintained between 19% and 24%, checked with an oximeter (WPI ISO2-D) in the chamber. After dissection of the eyes, cornea, iris, and lens were removed. The retina was dissected into four equal quadrants and attached photoreceptor surface down on a modified Biopore Millicell filter (Millipore). This preparation was transferred to a recording chamber and bathed (1 ml/min) with Ames. Pharmacological agents were also prepared in Ames. All experiments were performed at a near physiological temperature of 32 °C.
8
Retinal Electrophysiology in Mice
9
Retinal Preparation for Electrophysiology
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Mice retinae were prepared as previously described (Lefebvre et al., 2008 (link)). Briefly, mice were euthanized and retinae dissected out under infrared illumination to preserve dark adaptation. As much vitreous humor was removed as practical. A piece of retina was placed with ganglion cells facing down on an MSEA on the stage of an upright microscope. The preparation was continuously superfused with Ames' medium (A1420; Sigma Aldrich, St Louis, MO) equilibrated with 95% O2 and 5% CO2 gas at room temperature.
10
Retinal Flatmount Preparation
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The animal was deeply anesthetized with isoflurane and the first eye was enucleated. After the enucleation of the second eye, the animal was euthanized by CO2 inhalation. The retinal flatmounts were prepared as previously described (10 (link)). Briefly, an incision was made along the corneal limbus, the lens and sclera were removed, and radial cuts were made to relieve the curvature. The flat-mounted retina was transferred to a glass bottom dish (MatTek Corp.) and incubated at room temperature in the Ames’ medium (A1420, Sigma-Aldrich) oxygenated with 95%O2/5%CO2.
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