Detailed mappings of the vasculature were made so that the same vessels were studied throughout the experiment. Arterioles (45‐ to 60‐μm diameter and 0.5‐ to 1‐mm length and devoid of branching) were chosen, according to their visual clarity. The arterioles were occluded with a glass micropipette made with 1‐mm‐diameter glass tubing whose tip was drawn into a long fiber by a pipette puller (P‐87 horizontal puller; Sutter Instruments, Novato, CA). The fiber was bent over a flame, and the knee of the bend was pressed on the intact skin of the preparation mounted on an inverted microscope, which allowed observation of the opposite side of the window chamber (ie, the intact microcirculation) (23 (link); also see Figure 1 ). Arterioles were occluded for only 5 s, until flow completely stopped, and occlusion was released to prevent tissue hypoxia. Due to technical difficulties, only 4 to 6 experiments per mouse group could be analyzed. Additionally, the diameter was measured 200 μm from the occlusion point with recording of the occluded vessel starting 10 s prior to the occlusion, during the occlusion, and during 90 s after the reperfusion. The video image shearing technique was used to measure vessel diameter offline from video recordings.25 (link)
P 87 horizontal puller
Manufactured by Sutter Instruments
Sourced in United States
The P-87 horizontal puller is a piece of lab equipment used to pull and shape glass and quartz pipettes and needles. It operates by heating and stretching the material to produce the desired shape and size.
Automatically generated - may contain errors
5 protocols using p 87 horizontal puller
1
Mapping Microvascular Occlusion and Reperfusion
2
Xenopus Oocyte Microinjection Protocol
3
Dorsal Root Neurons Electrophysiology
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The experiments were performed at room temperature (20–22°C) in oxygenated solution containing (in mM): NaCl 125, KCl 2.5, CaCl2 2, MgCl2 1, NaH2PO4 1.25, NaHCO3 26 and glucose 10 (pH 7.4, 95% O2 and 5% CO2).
Dorsal root potentials and compound action potentials (CAPs) were recorded with a suction electrode from the L4 or L5 dorsal root close to its entrance to the spinal cord. The electrodes filled with the bath solution had a resistance of 20–100 kΩ.
Lamina I and X neurons were visualized for the whole cell patch-clamp recordings using the oblique infrared LED illumination technique (Safronov et al., 2007 (link); Szûcs et al., 2009 (link)). Patch pipettes pulled from borosilicate glass using a P-87 horizontal puller (Sutter Instruments, USA) had a resistance of 3–5 MΩ after filling with the solution of the following composition: 145 K-gluconate, 2.5 MgCl2, 10 HEPES, 2 Na2-ATP, 0.5 Na-GTP, and 0.5 EGTA (pH 7.3). Neurons were voltage clamped at −70 or −60 mV. Offset potentials were compensated before seal formation. Liquid junction potentials were not compensated.
MultiClamp 700B amplifier and Digidata 1320A/Digidata 1440 digitizers under the control of the pClamp software (Molecular Devices, CA, USA) were used for data acquisition. Signals were Bessel filtered at 2.6 kHz and sampled at 20 kHz. All chemicals were from Sigma-Aldrich (MO, USA).
Dorsal root potentials and compound action potentials (CAPs) were recorded with a suction electrode from the L4 or L5 dorsal root close to its entrance to the spinal cord. The electrodes filled with the bath solution had a resistance of 20–100 kΩ.
Lamina I and X neurons were visualized for the whole cell patch-clamp recordings using the oblique infrared LED illumination technique (Safronov et al., 2007 (link); Szûcs et al., 2009 (link)). Patch pipettes pulled from borosilicate glass using a P-87 horizontal puller (Sutter Instruments, USA) had a resistance of 3–5 MΩ after filling with the solution of the following composition: 145 K-gluconate, 2.5 MgCl2, 10 HEPES, 2 Na2-ATP, 0.5 Na-GTP, and 0.5 EGTA (pH 7.3). Neurons were voltage clamped at −70 or −60 mV. Offset potentials were compensated before seal formation. Liquid junction potentials were not compensated.
MultiClamp 700B amplifier and Digidata 1320A/Digidata 1440 digitizers under the control of the pClamp software (Molecular Devices, CA, USA) were used for data acquisition. Signals were Bessel filtered at 2.6 kHz and sampled at 20 kHz. All chemicals were from Sigma-Aldrich (MO, USA).
4
Electrophysiological Recording of Lamina X Neurons
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Electrophysiological recordings were made from lamina X neurons in ex vivo spinal cords in physiological bicarbonate solution. All recordings were made at room temperature. Patch pipettes were pulled from borosilicate glass using a P-87 horizontal puller (Sutter Instruments, Novato, CA, USA) and had a resistance of 3–5 MΩ when filled with intracellular solution containing (in mM) 145 K-gluconate, 2.5 MgCl2, 10 HEPES, 2 Na2-ATP, 0.5 Na-GTP and 0.5 EGTA (pH 7.3). For detecting genuine neuron firing activity, cell-attached recordings from the lamina X neurons were conducted after formation of a gigaseal. Whole-cell recordings in current and voltage-clamp modes were made after rupturing the patch of membrane. Electrophysiological signals were acquired and filtered (Bessel, 2.6 kHz) using MultiClamp 700B amplifier (Molecular Devices, Sunnyvale, CA, USA) and digitized with Digidata 1320A under control of pClamp 9.2 software (Molecular Devices, Sunnyvale, CA, USA).
5
Whole-cell Recordings of Spinal Lamina I Neurons
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The whole-cell recordings from labelled lamina I SPNs in the L4-L5 segments (Fig. 1Aa ) were performed at room temperature (20–22 °C) in oxygenated artificial cerebro-spinal fluid containing (in mM): NaCl 125, KCl 2.5, CaCl2 2, MgCl2 1, NaH2PO4 1.25, NaHCO3 26 and glucose 10 (pH 7.4, 95% O2 and 5% CO2). Patch pipettes were pulled from borosilicate glass using a P-87 horizontal puller (Sutter Instruments, USA); Pipette resistances were 3–5 MΩ when filled with intracellular solution containing (in mM): 145 K-gluconate, 2.5 MgCl2, 10 HEPES, 2 Na2-ATP, 0.5 Na-GTP and 0.5 EGTA (pH 7.3). Signals were acquired and filtered at 2.6 kHz using MultiClamp 700B amplifier (Molecular Devices, CA, USA) and digitized at 10 kHz with Digidata 1320 A under control of pClamp 9.2 software (Molecular Devices, CA, USA). Offset potentials were compensated before seal formation. Liquid junction potentials were not compensated.
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