scAAV8-hRS/IRBP-hRS vector was administered by intravitreal injection to pups at P5 or P6. This vector is comparable to the clinical vector in a human XLRS trial [14 (link)]. Pups were anesthetized either by 20–30 µL volume via intraperitoneal injections of ketamine/xylazine mixture containing ketamine hydrochloride (30–40 mg/kg) and xylazine (3–4 mg/kg) for 10–15 min or by isoflurane inhalation (1.5% isoflurane in 100% oxygen) for 3 to 5 min. One drop of 0.5% tetracaine topical anesthetic was applied, and pupils were dilated using a drop each of 1% tropicamide and 2.5% phenylephrine. One drop of Proparacaine hydrochloride 0.5% was applied as topical ocular anesthesia. A small scleral nick was made at the nasal-temporal ora serrata below the iris using a 30-G insulin syringe needle to gain access to the posterior cavity (vitreous chamber) of the eye. Sterile 10 µL Nanofil syringes with the 35 G beveled-tip needle on a sterile 10 µL Nanofil syringe (World Precision Instruments, Inc., Sarasota, FL, USA) was inserted into the vitreous to deliver 2e10 (2 × 1010) vector genomes per eye (vg/eye) in 1.5 μL fluid. The contralateral eye served as control. Triple antibiotic ophthalmic ointment (neomycin, polymyxin B, and bacitracin) was applied to the eye at the conclusion. Animals were kept on a warming pad until their recovery [15 (link)]
Nanofil syringe
Manufactured by World Precision Instruments
Sourced in United States
The Nanofil syringe is a precision instrument designed for accurate and controlled fluid delivery. It features a small-diameter tip for handling micro-volumes. The syringe is constructed with high-quality materials and components to ensure reliable and consistent performance.
Automatically generated - may contain errors
Lab products found in correlation
Ultramicropump 3, by World Precision Instruments (6 mentions)
Operating microscope, by Leica (6 mentions)
33 g blunt needle, by World Precision Instruments (5 mentions)
33g beveled needle, by World Precision Instruments (3 mentions)
Avertin, by Merck Group (3 mentions)
Ketamine, by Vedco (3 mentions)
Xylazine, by Vedco (3 mentions)
Acepromazine, by Vedco (3 mentions)
Nanofil needle, by World Precision Instruments (3 mentions)
Sys micro4 controller, by World Precision Instruments (2 mentions)
Stereotaxic frame, by Kopf Instruments (2 mentions)
Genteal, by Novartis (2 mentions)
Nano pump controller, by KD Scientific (2 mentions)
Removable 35 gauge needle, by World Precision Instruments (2 mentions)
33 gauge needle, by World Precision Instruments (2 mentions)
33 gauge blunt needle, by World Precision Instruments (2 mentions)
Nf35bv 2, by World Precision Instruments (2 mentions)
35g blunt ended needle, by World Precision Instruments (2 mentions)
C57bl 6j mice, by Jackson ImmunoResearch (2 mentions)
Microsyringe pump controller, by World Precision Instruments (2 mentions)
110 protocols using nanofil syringe
1
Intravitreal Injection of scAAV8-hRS/IRBP-hRS Vector
2
Inhibiting mTOR to Treat Laser-Induced CNV
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All recombinant AAV vectors were derived from scAAV2 vectors. The mTOR siRNA (5′-GAAUGUUGACCAAUGCUAU-3′) was designed from the completely conserved multi-species region found in humans (NM_004958), monkeys (XR_014791), rats (NM_019906), and mice (NM_020009) to establish rAAV-mTOR shRNA-EGFP. A scrambled control siRNA (5′-AUUCUAUCACUAGCGUGAC-3′) was prepared to make rAAV-scrambled control shRNA-EGFP (rAAV-scrambled shRNA-EGFP). Both of the scAAV2 vectors use the H1 promoter to express either mTOR siRNA or the scrambled control siRNA, whereas EGFP expression is driven by the cytomegalovirus promoter. All rAAV vectors were supplied by CdmoGen. Intravitreal injections of the vector were performed in the right eyes of the mice, with pupil dilation, 5 days after laser photocoagulation under anesthesia using 35G blunt needles with Nanofil syringes (World Precision Instruments). One microliter of the viral vectors at a concentration of 5.0 × 1010 viral genomes (vg)/mL was used per injection. Laser photocoagulation was used to induce CNV in three groups of mice (n = 20 per each group) before being injected with the following: 0.1% PBS for the first group, rAAV-scrambled shRNA for the second, and rAAV-mTOR shRNA for the third. As a negative control, 10 mice were not treated with laser photocoagulation or intravitreal injections. Five mice from each group were used for qRT-PCR.
3
Bilateral Injection of AAV into aIC
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1.0 µl of AAV solution was injected bilaterally into aIC (AP: +2.8, ML: +4.0, DV: −5.9 mm from Bregma) over 5 min using 10 µl Nanofil syringes (World Precision Instruments), with 33-gauge needles, attached to a UltraMicroPump (UMP3) with SYS-Micro4 Controller (World Precision Instruments). The needle was left in place for an additional 5 min.
4
Striatal Injection: Minimally Invasive Technique
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Mice were anesthetized with isoflurane and positioned on a stereotaxic frame (David-Kopf Instruments, Tujunga, CA). A 1-cm midline incision was made in the skin over the dorsal surface of the skull, and the skull was exposed to allow the positioning of a drill over the bregma point of reference. From the bregma, the coordinates used were +1 mm anteroposterior, +1.7 mm lateral, and −3 mm ventral to the skull surface. These coordinates were selected to target the center of the left striatum and avoid passing through any ventricle. BHs were injected with Nanofil syringes (World Precision Instruments, Sarasota, FL) and steel bevel needles (33-gauge diameter; World Precision Instruments) into the striatum at a rate of 0.25 µl/min with a total of 0.5 µl per mouse controlled with a pump (UltraMicroPump III with a Micro4 pump controller; World Precision Instruments). The needle was kept in place for 2 min following injection to avoid any reflux of the BH solution. The skin incision was closed with sutures. These conditions produced minimal mechanical trauma to the brain. The patency of the needles was verified prior to and after injections. Mice were euthanized by isoflurane anesthesia overdose, followed by cervical dislocation, at different times after injection.
5
Targeted AAV8 Delivery to Anterior Insular Cortex
6
Intravitreal AAV Gene Delivery in CNV
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Five days after laser photocoagulation, AAV was injected intravitreally into the right eye under anesthesia with pupil dilation, using a 30G sharp needle and a 35G blunt needle fitted with NanoFil syringes (World Precision Instruments, Sarasota, FL). A sclerotomy was created approximately 0.5 mm posterior to the limbus using a sharp 30G needle tip prior to vector administration. One microliter of AAV2-EGFP, AAV5-EGFP, and AAV8-EGFP (1 × 1013 viral particles [vp]/ml) supplied by CdmoGen (Cheongju, Republic of Korea) were used for injection. Intravitreal injection was performed using a syringe fitted with a 35G blunt needle while visualizing the fundus directly using a surgical microscope and a small plastic ring filled with 0.5% methylcellulose on the cornea (GenTeal; Novartis, Basel, Switzerland). Ten and five mice with and without CNV, respectively, were injected with each AAV serotype; thus, a total of 45 mice underwent intravitreal vector administration.
7
Adenoviral Vector-Mediated Gene Delivery
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Ad5mK8-nlsCre (VVC-Li-535; University of Iowa Viral Vector Core) was diluted in DMEM with CaCl2 (10 mM) and introduced into the fourth mammary ducts at a titer of ~1.92 × 107 pfu/duct using 100 µL Nanofil syringes (World Precision Instruments) fitted with 35 G Nanofil needles (World Precision Instruments). Live animal and organ imaging was performed using an IVIS Spectrum imaging system (Perkin Elmer). Imaging of lung metastases was performed on a Zeiss Axio Observer.Z1 microscope with Zeiss ZEN 3.3 Blue software.
8
Monitoring Virus Replication during Larval-Adult Transition
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To monitor virus replication during the transition of larvae into adults, freshly deposited third instar larvae were injected with either PBS or virus suspension using a modified protocol previously described by Jura et al. (1993) (link). Briefly, larvae were immobilized at 4°C for 1 min and then injected with 1 μl of PBS or virus suspension using a 100-μl NanoFil syringe (World Precision Instruments, Inc., Sarasota, FL, United States) equipped with a 35-gauge beveled needle. The needle was accurately placed 1 mm away from the two larval polypneustic lobes. Correct injection was verified by observing blanching of the larva. Larvae were then placed in plastic dishes over ice for 1 min to allow wound-healing and subsequently allowed to pupate (in this manuscript pupate and pupation refer to pupariate and pupariation) for 2 h at room temperature. Successful pupation rates were assessed 24 h post larval-injection; pupae were incubated at 25 ± 0.5°C until adult emergence. Ten days post adult eclosion, all flies were assessed for the occurrence of SGH symptoms by microscopy during SG dissections. The total DNA was extracted from the fly carcasses, and the SGs were homogenized in PBS (1 pair of SG/100 μl PBS) and assayed for virus presence by PCR (Abd-Alla et al., 2007 (link)).
9
Chemogenetic Manipulation of CeA Neurons
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PKC-δ-Cre or SOM-Cre mice of 2–3 months old of either sex were injected with viruses in the CeA for chemogenetic studies. Mice were deeply anesthetized with isoflurane (4% induction, 1.5%–2% maintenance in oxygen, vol/vol; Halocarbon Laboratories, USA) and positioned in a stereotaxic injection frame (IVM-3000; Scientifica, Uckfield, UK). The coordinates of bilateral injection sites of the CeA were AP: − 1.31 mm, ML: ±2.87 mm, DV: − 4.82 mm. During all surgical procedures, mice were kept on a homeothermic pad (Physitemp Instrument, USA or TMP-5b, Supertech Instruments, Hungary) to maintain their body temperature at 34–37 °C. After securing the head with ear bars, the eyes of the mice were protected by the ophthalmic gel. For the virus injection, 0.35 μl/side of virus were bilaterally injected into the CeA using a 10 μl NanoFil syringe (World Precision Instruments, USA) and a 34-G beveled metal needle, controlled by the nano-pump controller (0.1 μl/min, KD Scientific, USA). After 10 min for virus distribution, the needle was withdrawn slowly. All animals were rendered to recover for at least 3 weeks before the behavioral tests for maximal viral expression.
10
Retrograde Tracing of Central Circuits
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For retrograde labeling the target circuits in the central nervous system, we bilaterally injected 0.35 μl CTB-594 (C22842, Thermo, Invitrogen) into CeA using a 10 μl NanoFil syringe (World Precision Instruments, USA) and a 34-G beveled metal needle, controlled by a nano-pump controller (0.1 μl/min, KD Scientific, USA). After 10 min for CTB-594 distribution, the needle was withdrawn slowly. All animals were allowed to recover for at least 1 week before the behavioral tests.
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