Mice were placed in a stereotaxic apparatus (RWD Life Science, San Diego, CA, USA) under isoflurane (3%, inhalation) anesthesia, and the skull was exposed. A small hole was then made using a dental drill. Virus was bilaterally injected into the nucleus accumbens core (from the bregma: AP + 1.4 mm, ML ± 1.5 mm, DV -3.6 mm from the brain surface at an angle of 10°: 1.0 μL was applied to each side using a Hamilton syringe), the lateral shell (from the bregma: AP + 1.0 mm, ML ± 1.8 mm, DV -4.9 mm from the skull at an angle of 0°: 300 nL was applied to each side using a Nanoject III (Drummond Scientific Company, Broomall, PA, USA)) and the medial shell (from the bregma: AP + 1.5 mm, ML ± 0.5 mm, DV − 4.7 mm from the skull at an angle of 0°: 300 nL was applied to each side using a Nanoject III (Drummond Scientific Company)).
Nanoject 3
Manufactured by Drummond
Sourced in United States
The Nanoject III is a microinjection system designed for precise and controlled delivery of small volumes of liquids into cells or tissues. The device features an automated microinjector that allows for programmable injection parameters and supports a range of sample volumes from nanoliters to microliters. The Nanoject III is compatible with a variety of sample types and can be used in a wide range of applications requiring accurate liquid handling.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Infusion cannula, by RWD Life Science (3 mentions)
Nanoject 2, by Drummond (3 mentions)
Stereotaxic frame, by Kopf Instruments (3 mentions)
Stereotaxic apparatus, by Kopf Instruments (3 mentions)
Round glass coverslip, by Harvard Apparatus (2 mentions)
Alexa 647, by Thermo Fisher Scientific (2 mentions)
C3667, by Merck Group (2 mentions)
Induction chamber, by Baxter (2 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (2 mentions)
Aav flex tdtomato, by Addgene (2 mentions)
Carprofen, by Zoetis (2 mentions)
Mmessage mmachine t7 transcription kit, by Thermo Fisher Scientific (2 mentions)
Rneasy minelute cleanup kit, by Qiagen (2 mentions)
Minelute pcr purification kit, by Qiagen (2 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (2 mentions)
Aav ef1a doubled floxed hchr2 h134r mcherry, by Addgene (2 mentions)
Aav ef1a doubled floxed hchr2 h134r eyfp, by Addgene (2 mentions)
Desipramine, by Merck Group (2 mentions)
Wiretrol 2, by Drummond (2 mentions)
146 protocols using nanoject 3
1
Viral Targeting of Nucleus Accumbens Subregions
2
Stereotactic Viral Transduction of Striatal Neurons
3
Stereotactic AAV Injection in Mice
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We performed AAVs injection as previously described52 (link),53 (link). In brief, mice 6–8 weeks of age were stereotaxically injected. Animals were anesthetized with isoflurane (1–2%) and placed in a stereotaxic frame. The adeno-associated virus AAVDJ-CaMKII-iCre-T2A-mCherry (3.55~7.87 ×1011 vg/mL) or AAVDJ-CAMKII-mCherry (7.83 × 1012 vg/mL) were injected to the olfactory bulb or the PC bilaterally. For AAV transduction to the granule cell layer of the olfactory bulb, we injected 300 nL of AAV to each olfactory bulb bilaterally. For AAV transduction to the PC, we injected AAV to three coordinates to either side of the brain, with a total of six injection coordinates bilaterally with 300 nL injection volume per injection coordinate. For injection, we used a pulled glass capillary (Drummond) and injected through nanoliter pressure injection (Nanoject3, Drummond). Stereotactic injection coordinates to target the olfactory bulb or PC were obtained from the Paxinos and Franklin atlas (for olfactory bulb AP:4.2 mm ML:1.0 mm DV:1.0 mm from brain surface; for PC AP:1.8, 0.5, −0.5 mm ML:2.7, 3.5, 3.9 mm, DV:3.8, 3.8, 4.2 mm from brain surface). Animals were allowed to recover for at least 4 weeks before being used in experiments.
4
Viral Manipulation of vCA1 and BLA in Mice
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For mice that underwent chemogenetic manipulations, adult males (8–9 weeks old) received viral micro-infusion in the same surgery as head bar attachment, similar to previously described protocol 46 (link). Specifically, AAV8-hSyn-DIO-hM3D(Gq)-mCherry (Addgene, 44361-AAV8, 2.9 × 1013 vg/ml) or AAV8-hSyn-DIO- mCherry (Addgene, 50459-AAV8, 1.0 × 1013 vg/ml) was micro-infused in vCA1 bilaterally (500 nL per hemisphere, −3.52 mm AP, ±3.1 ML, −4.2 [150 nL], −4.1 [200 nL] and −4.0 [150 nL] DV, from bregma according to Paxinos and Franklin [2011]), and AAV2retro-CAG-Cre (UNC Vector Core, Ed Boyden’s stock, 4.1×1012 vg/ml) was micro-infused in BLA bilaterally (500 nL per hemisphere, −1.80 mm AP, ±3.1 ML, −5.0 [150 nL], −4.8 [200 nL] and −4.6 [150 nL] DV). Viral vectors were delivered using Nanoject 3 (Drummond Scientific, Broomall, PA). The needle was held in place for >5 min after infusion at each DV, and for 10 min after the last DV. Following viral micro-infusion, head bar was attached to the skull as described above.
5
Virus Injection in Mouse Brain
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Glass capillaries (OD 1.14 mm: ID 0.53 mm) were pulled and beveled at a 40-degree angle36 (link), and mounted in a NanoJect 3 (Drummond Scientific, USA). The pipette was front-loaded with the virus solution and 150nL injected at a depth of 350–500 µm below the dura, in 5 nL steps. After the last injection, the pipette was left in the tissue for five minutes before retraction and loading of a new pipette. Two to three different constructs were injected per animal, spaced at least 700 µm apart. After the final injection, the exposed brain surface was cleaned with saline, and a cranial window implanted as described above.
6
Cre-Dependent Viral Injections and GCaMP Imaging
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For virus injections, the same procedure was followed as described in Ref.20 (link). Briefly, Ntsr1-cre mice (10–15 weeks old) were anaesthetized under isoflurane (2%–5%) and craniotomies performed. Cre-dependent AAV viruses encoding the fluorescent protein GFP were injected in layer 6 of the primary visual cortex (at a depth of 900 µm). After injections, the craniotomy was sealed with silicon (kwik-cast) and animals were allowed to recover for 2–3 weeks. Viruses were delivered at a rate of 1–2 nl/s using Nanoject III (Drummond Scientific, USA).
For in vivo GCaMP7f imaging, mice were anaesthetized with isoflurane (2%–5%) and implanted with a headplate. A craniotomy (3 mm) was drilled over the primary visual cortex, and 100 nl of a virus encoding GCaMP7f (AAV1-syn-jGCaMP7f-WPRE, titre: 4.75 × 1012 ml−1) was injected in layer 2/3 of the primary visual cortex (Bregma: − 2.7 mm, ML: − 2.58 mm, depth: 0.25 mm) at a rate of 1 nl/s. The craniotomy was sealed by implanting a 3 mm diameter round glass coverslip (#1, Harvard Apparatus, USA) to create a chronic cranial window.
For in vivo GCaMP7f imaging, mice were anaesthetized with isoflurane (2%–5%) and implanted with a headplate. A craniotomy (3 mm) was drilled over the primary visual cortex, and 100 nl of a virus encoding GCaMP7f (AAV1-syn-jGCaMP7f-WPRE, titre: 4.75 × 1012 ml−1) was injected in layer 2/3 of the primary visual cortex (Bregma: − 2.7 mm, ML: − 2.58 mm, depth: 0.25 mm) at a rate of 1 nl/s. The craniotomy was sealed by implanting a 3 mm diameter round glass coverslip (#1, Harvard Apparatus, USA) to create a chronic cranial window.
7
Optogenetic Manipulation in Rodent Barrel Cortex
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All viral vectors were administered into the right barrel cortex (coordinates relative to Bregma: 1.5 mm posterior, 3.3 mm lateral, and 0.5 mm deep) using a Nanoject III apparatus (Drummond Scientific, Broomall, PA) under isoflurane anesthesia (2%–3%). For the optogenetics experiments, we injected 500 nl of AAV1-CAGGS-FLEX-rev-ChR2-tdTomato (1 × 1013 genomic copies per mL [82 (link)]) and installed a custom-made headpost using dental cement. Three weeks post injection, we made a 1-mm–diameter craniotomy (leaving the dura intact) near the injection site. The exposed brain was kept moisturized throughout the surgery (and later, the experiment) with the following extracellular solution (in mM): 150 NaCl, 2.5 KCl, 10 HEPES, 2 CaCl2, 1 MgCl2 (pH 7.3, adjusted with HCl/NaOH; 300 mOsm).
8
Adult Fly Thorax Injection Protocol
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500 cells/10 nL/fly were injected into the thorax of CO2-anesthetized adult male flies using a nanoliter injector (Nanoject III, Drummond). The injected flies were raised at 25°C up to 11 days after injection.
9
Viral Vector Injections into Auditory Cortex
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For all animals, bilateral surgical virus injections into the AC were performed on P23 or P24, after earplug removal. AAV1.hSyn.eGFP.WPRE.bG (5×1012 vg/mL) was used as a control. Animals were anesthetized with isoflurane, and incisions made just ventral to the temporal ridge. A small burr hole (0.7 mm diameter) was drilled in the skull ~1.4 mm below the temporal ridge, just over the AC (Radtke-Schuller et al., 2016 ). Virus was loaded into a glass pipette backfilled with mineral oil. The glass pipettes were made sharp enough to penetrate dura without causing damage and were inserted to a depth of 400 μm. Virus was injected at a rate of 2 nl/s with a Nanoject III (Drummond), followed by a 5 min period to permit diffusion. For the two custom viruses, a volume of 600 μl was injected, whereas for the control GFP virus, a volume of 250 μl was injected. Incisions were closed with surgical adhesive and the animal permitted to recover for 7 days.
10
Ae. aegypti Mosquito Immune Response to EVs
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Four to five-day old Ae. aegypti (Liverpool strain) female mosquitoes were intrathoracically injected with 69 nL of LPS (1 mg/mL) [Sigma Aldrich] or 1X dPBS (Thermo Fisher Scientific) using a Nanoject III injector (Drummond Scientific Company, Broomall, PA, USA) and incubated for six h at 27 °C prior to EV injection. Mosquitoes were then challenged with serial dilutions of 1 × 107, 1 × 106, 1 × 105 EVs, or 1X dPBS as a control. Total RNA was isolated from three biologically distinct batches of 8 mosquitoes per treatment group 24 h post-challenge. Mosquitoes were homogenized using a mortar and pestle in 1 mL of Trizol. The resulting suspension was centrifuged at 12,000 × g for 10 min at 4 °C to remove debris, and the supernatant collected. RNA extraction, cDNA synthesis and qPCR were performed as described above.
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