Microinjection needles were pulled on a horizontal micropipette puller (Flaming/Brown P-97, Sutter Instruments, Novato, CA, USA) using fire-polished, filamentous borosilicate glass (outer diameter of 1.2 mm, Sutter Instruments). Morpholino solutions were prepared fresh prior to the microinjection procedure. Control MO was injected at 0.25–1 mM in 0.1% phenol red (Sigma) and 125 ng/μL rhodamine-conjugated dextran. Fluorescently tagged α2A MOs were injected at 0.1–2 mM in 0.1% phenol red. Each solution was loaded into a micropipette needle and was then injected in the yolk stream of 1–2 cell stage zebrafish embryos. Following microinjection of the morpholinos, the embryos were individually screened at 5–6 hpf looking for fluorescence at the animal pole using epi-fluorescence and a 10x objective on an inverted microscope (Zeiss Axiovert 200M) using minimum light intensity (See Supplement Fig. 1A ). Embryos lacking fluorescence were removed and thus not used in subsequent experiments.
P 97 flaming brown
Manufactured by Sutter Instruments
Sourced in United States
The P-97 Flaming/Brown is a micropipette puller designed for producing high-quality micropipettes. It utilizes a high-heat, P-97 filament heating system to pull and shape glass capillaries into precise micropipette tips.
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Lab products found in correlation
6 protocols using p 97 flaming brown
1
Microinjection of Morpholino Oligos in Zebrafish
2
Patch Clamp of Myenteric Neurons
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Jejunal tissue was prepared for patch clamp of myenteric neurons using hemidissection as described previously24 (link). Patch pipettes were pulled on a Flaming-Brown-P97 (Sutter Instruments, Novato, USA) electrode puller to produce 4–7 MΩ micropipettes. Signals were 4 point Bessel filtered at 2 or 5 kHz, then digitized to 5 or 20 kHz. Positive pressure was applied to the pipette as the tip entered solution until contact with the neuron was made. Whole cell recording mode began after suction and the amplifier was then switched to current-clamp mode. Recordings were filtered such that only those with resistances >4 GΩ were used. Epithelial stimulation and recordings were made as described previously24 (link), using Multiclamp 700B Amplifier, Digidata 1440A signal converter and PClamp 10.7.0 software (Molecular Devices). Neuron excitability, AP, and membrane properties were assessed first with Krebs buffer in the epithelial compartment (~20 min) and then with inflow to the chamber switched to a solution containing the drug treatment. Patch clamp experiments had an n = 8/group.
3
Ca2+ Puffing and Imaging in DGCs
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Borosilicate glass pipettes (with a tip diameter of 5 μm and resistance of 1 MΩ when filled with ACSF) were fabricated on a micropipette puller (P-97 Flaming/Brown, Sutter Instruments), backfilled with 100–250 mM CaCl2 and placed into a pipette holder mounted on a motorized micromanipulator with a closed pressure system connected to a 1 mL syringe. The pipette tip was placed adjacent to groups of DGCs expressing Peredox and RCaMP biosensors. Pressure delivery was controlled by a solenoid valve (NResearch) in series with the syringe line that was triggered by a digital/analog actuator (NTE Electronics). Positive pressure was applied using the syringe, and Ca2+ puffs were delivered by opening the solenoid for 0.5–5 s beginning 300 ms after the start of image acquisition.
4
Zebrafish Xenograft Model for Pancreatic Cancer
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For zebrafish xenografts, wild type Tübingen (TU) zebrafish were bred and maintained in the Western Australian Zebrafish Experimental Research Centre (Biomedical Research Facility- Shenton Park, Western Australia). Experiments and data analyses were done as previously described [32 (link)]. Briefly, HPAF-II human pancreatic cancer cells were incubated with Vibrant™-Dil dye (ThermoFisher Scientific) 4 μL/mL in HBSS at 37 °C for 10 min, followed by 15 min on ice in the dark. Cells were then harvested and resuspended at a density of 107 cells/, loaded into a capillary glass needle using a puller (p-97 Flaming/Brown by Sutter Instrument®) and 10 nL of cell suspension (approximately 100 cells/embryo) was injected in the perivitelline space of 24-h post fertilisation (hpf) zebrafish embryos. Zebrafish were incubated at 34 °C O/N to allow for cell growth and the following day embryos were equally distributed in to three treatment groups. One group did not receive any treatment or cells (blank), a second group was treated with 0.1% DMSO and a third group was treated with 10 μM JVG045. At 5 days post fertilisation (dpf), the effect of the drugs on cancer cell growth was documented using a fluorescent stereomicroscope equipped with a digital camera (Nikon SMZ Zoom). Images were analysed using ImageJ. Non injected embryos were used to subtract background fluorescence.
5
Micropipette Fabrication from Glass Capillaries
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Glass capillaries [1.2 mm O.D. × 0.94 mm I.D., Harvard Apparatus (BF‐120‐94‐10)] were pulled into micropipettes using a micropipette puller (P‐97 Flaming Brown, Sutter instruments, Novato, CA). See User Manual and guidelines therein.
6
Electrochemical Characterization at Liquid-Liquid Interface
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Cyclic voltammetric experiments were performed using a three-and four-electrode configuration with an Autolab PGSTAT100 potentiostat (Metrohm-Autolab). iR compensation was applied for the electrochemical measurements. Homemade Ag/AgCl reference electrodes (RE) were directly immersed in the chloride-containing aqueous phase, an aqueous solution of 0.1 mM LiCl and 1 mM BTPPACl was brought in contact with the organic solution via a micropipette and formed a liquid junction for the organic reference electrode. The micropipette was prepared from a borosilicate capillary (o.d. 1.5 mm, i.d. 1.1 mm), using a micropipette puller (Sutter P-97 Flaming/Brown). The average internal diameter of the micropipette tip was estimated to be 2-3 µm. The aqueous counter electrode (CE w ) was glass coated to avoid contact of the Pt with the organic (upper) phase (all metals were obtained from Advent Research Materials). The cells used for the liquid/ liquid electrochemical measurements at the organic/water interface, had a working area in the range 0.74-0.83 cm 2 and a total solution volume of 2.5 mL. The errors presented are either standard deviations (arithmetic averages of multiple measured values) or absolute errors determined from the best fit errors.
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