Animals were anesthetized with 3 ml/kg equithesin (a combination of sodium pentobarbital, chloral hydrate, ethanol, and magnesium sulfate, all from Sigma-Aldrich, St. Louis, MO) and placed into a Kopf stereotaxic frame (Tujunga, CA). For all experimental groups, rAAV5-hSyn-ChR2(H134R)-mCherry (UNC Vector Core) was microinjected into the DLSC bilaterally through a 30-gauge dental needle. This vector allows for neuron-specific targeting of opsin expression (Kügler et al., 2003 (link)). The injection coordinates for the DLSC were: 5.0 mm posterior to bregma, 2.5 mm lateral to midline, and 4.5 mm ventral to the dura, with the incisor bar 5.0 mm above the interaural line (Pellegrino and Cushman, 1967 ). Microinjections consisted of 1.5–2μl of virus that was injected sequentially in each DLSC at a rate of 0.2 μl/min; the injection needle was left in place for at least 5 min to allow virus diffusion before retraction. Following the microinjections, an optical fiber (200 μm core, 0.22NA, Thorlabs, Newton, NJ) was implanted 0.2 mm dorsal to each injection site. optical fibers were made according to a previously described protocol (Sparta et al., 2012 (link)) and held in place with jeweler’s screws and dental acrylic.
Equithesin
Manufactured by Merck Group
Sourced in United States
Equithesin is a laboratory equipment product manufactured by Merck Group. It is designed to facilitate certain laboratory procedures. The core function of Equithesin is to assist in the given tasks within the laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
Ketorolac, by Merck Group (3 mentions)
Infusion harness, by Instech (3 mentions)
Ketamine hydrochloride, by Vedco (2 mentions)
Optical fiber, by Thorlabs (1 mentions)
Ketamine, by Vedco (1 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Chloral hydrate, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium f12, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Stereotaxic frame, by Kopf Instruments (1 mentions)
7 protocols using equithesin
1
Optogenetic manipulation of DLSC neurons
2
Surgical Implantation for Cocaine Self-Administration
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For cocaine self-administration and yoked saline controls, rats underwent catheter implantation 5 to 7 days before the start of self-administration as previously described (Zhou et al., 2012 (link)). Briefly, rats were anesthetized (IP) using a mixture of ketamine hydrochloride (66mg/kg; Vedco Inc., St. Joseph, MO) and xylazine (1.33mg/kg; Lloyd Laboratories, Shenandoah, IA), followed by Equithesin (0.5mL/kg) and Ketorolac (2mg/kg; Sigma Aldrich) as a preoperative analgesic. One end of a silastic catheter was implanted into the right jugular vein, and the other end was attached to an infusion harness (Instech Solomon, Plymouth Meeting, PA) for IV drug delivery.
3
Cocaine Self-Administration in Rats
4
Sciatic Nerve Crush Injury in Rats
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A total of 30 female Sprague-Dawley rats (age, 2 months; weight, 180–220 g) were purchased from The Experimental Animal Center of Nantong University (Nantong, China) and subjected to sciatic nerve crush injury as previously described (11 (link)). Rats were anaesthetized by injecting 2 ml/kg Equithesin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), as previously described (12 (link)), corresponding to 17 mg/kg sodium pentobarbital, 42 mg/kg magnesium sulfate and 85 mg/kg trichloroacetaldehyde monohydrate) (11 (link),16 (link)). Anesthesia was assessed by loss of pedal reflexes. Rat left sciatic nerves were crushed with forceps for 10 sec, three times. To minimize the discomfort and possible painful mechanical stimulation, the rats were housed in large cages with sawdust bedding post-surgery and maintained in a controlled environment under constant temperature of 25°C and relative humidity of 40–70%, with a 12 h light/dark cycle and free access to food and water. Rats were sacrificed 24 h, 4 days, or 1, 2, 3, 4 or 8 weeks following surgery. Gastrocnemius muscles from the left (injured) side and the right (uninjured) side were collected. Control rats were anesthetized without sciatic nerve crushing (sham surgery). All animal procedures were ethically approved by The Administration Committee of Experimental Animals of The Jiangsu Province (Jiangsu, China).
5
Surgical Implantation of Jugular Catheter in Rats
6
Isolation and Culture of Neural Stem Cells
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NSCs were obtained from the embryos on embryonic day (E)15, as described previously (He et al., 2018). In brief, after intraperitoneal anaesthetization with 2 mL/kg equithesin (pentobarbital sodium, Cat# Y0002194, Sigma-Aldrich, St. Louis, MO, USA; chloral hydrate, Cat# 15307, Sigma-Aldrich), the embryos were removed by cesarean section, and the embryonic hippocampi were isolated. The hippocampi were then combined and rapidly converted into a single-cell suspension by mechanical dissociation. The cell suspensions were maintained in cell culture flasks in Dulbecco's modified Eagle's medium/F12 (1:1; Gibco, Grand Island, NY, USA) containing 2% B27 (Gibco) supplemented with epidermal growth factor (20 ng/mL; Sigma-Aldrich) and basic fibroblast growth factor (20 ng/mL; Sigma-Aldrich). Cells were incubated at 37˚C 5% CO2 and passaged every 5 days by dissociating the newly formed neurospheres with trypsin (Gibco). The NSCs were then randomly divided into miR-NC, miR-103-3p, LV-NC, LV-Ndel1, and LV-Ndel1 + miR-103-3p groups.
7
Intracerebroventricular Infusion of Amyloid-Beta 1-42 in Rats
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The Aβ 1-42 peptide was purchased from Tocris (Bristol, UK) and was freshly prepared in sterile double-distilled water (vehicle) at a concentration of 4 μM as previously described [15] . Seven-weeks-old rats were anaesthetized with 3.6 ml/kg Equithesin intraperitoneally (i.p.; composition: 1.2 g sodium pentobarbital; 5.3 g chloral hydrate; 2.7 g MgSO4; 49.5 ml propylene glycol; 12.5 ml ethanol and 58 ml distilled water) (Sigma Aldrich. Milan Italy) and secured in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). Coordinates from bregma for intracerebroventricular (icv) infusions were based on the atlas of Paxinos and Watson [69] : AP = -0.5, ML = +1.2 and DV = -3.2, (incisor bar at -3.3 mm). Soluble Aβ (5 μl) was delivered through a 25 μl Hamilton microsyringe at 2 μl/min infusion rate over a period of 2.5 min. Infusion needle was left in place for additional 5 min in order to avoid elapsing during removal. Control rats received vehicle only, because outcomes observed from preliminary used reverse Aβ 42-1 were indistinguishable from vehicle alone (unpublished observations). The needle track placement of was verified at the time of dissection. All experimental procedures were carried out 7 days after icv administration (SHAM or Aβ-treated groups).
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