Mice were anesthetized (induction: 5%, maintenance 0.5% isoflurane in O2 vol/vol). Following anesthetization, mice were attached to a stereotactic apparatus and the right hemisphere of the dentate gyrus (DG) was injected with 1 μL of a DJ‐serotype AAV vector encoding the red‐shifted calcium indicator jRGECO1a under the CamKII promoter at 1012 GC/mL titer. Viral injections were done with a pulled glass pipette using a Nanoject III microinjector (Drummond). The virus was injected at 33 nL per cycle for 31 cycles at a rate of 10 nL/s with a 20 s delay between cycles, followed by a 15 min wait period before the pipette was removed. The location of the viral injection was determined by first finding the midpoint between bregma and lambda. The mediolateral coordinate was 1.5–1.75 mm from the the lambda‐bregma midpoint and the dorsoventral coordinate was 1.8–2.0 mm from the dura, depending on bregmalambda distance, which ranged from 3 to 4 mm (Table S1 ).
Nanoject 3 microinjector
Manufactured by Drummond
The Nanoject III microinjector is a precision instrument designed for the accurate delivery of nanoliter volumes of liquids into cells, tissues, or other small-scale applications. It features a microprocessor-controlled stepper motor that allows for the precise and repeatable injection of volumes ranging from 0.46 to 69 nL per injection. The Nanoject III is a versatile tool suitable for a variety of research and laboratory applications that require the controlled and consistent delivery of small liquid volumes.
Automatically generated - may contain errors
8 protocols using nanoject 3 microinjector
1
Calcium Imaging of Dentate Gyrus
2
RNA Interference in Mosquitoes
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For RNAi in adult mosquitoes, 20 cold-anesthetized 3-day-old female mosquitoes were injected with 69 nl dsRNA solution of Met or EcR (3 μg/μL) into the hemocoel using Nanoject III microinjector (Drummond). Mosquitoes injected with dsGFP were used as negative controls. RNAi efficacy was examined at 3 days postinjection.
For RNAi in mosquito larvae, groups of 50 new hatched first-instar larvae were reared separately in 90 mm petri dishes, and dsRNA of Met or EcR was supplied into water at final concentration of 10 μg/mL. dsGFP was used as negative control. RNAi efficacy was tested at 7 days post-treatment, and mortality was recorded daily.
For RNAi in mosquito larvae, groups of 50 new hatched first-instar larvae were reared separately in 90 mm petri dishes, and dsRNA of Met or EcR was supplied into water at final concentration of 10 μg/mL. dsGFP was used as negative control. RNAi efficacy was tested at 7 days post-treatment, and mortality was recorded daily.
3
Microinjection Protocol for Vascular and Lymphatic Labeling
4
Calcium Imaging in Mouse Dentate Gyrus
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Mice were anesthetized (induction: 5%, maintenance 0.5% isoflurane in O2 vol/vol). Following anesthetization, mice were attached to a stereotactic apparatus and the right hemisphere of the dentate gyrus (DG) was injected with 1μl of a DJ-serotype AAV vector encoding the red-shifted calcium indicator jRGECO1a (25 (link)) under the CamKII promoter at 1012 GC/ml titer. Viral injections were done with a pulled glass pipette using a Nanoject III microinjector (Drummond) at previously described coordinates (51 (link)).
5
Knockdown of Aedes aegypti genes
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A total of 400 ng dsRNA was injected into each adult female Ae. aegypti using a Nanoject III microinjector (Drummond) with a glass needle drawn from a capillary. For each dsRNA, 3 replicates were performed with ~ 30 females per replicate. A pooled dsRNA group was also performed (400 ng/dsRNA) with the genes AAEL000417, AAEL003318, AAEL004513 and AAEL012440 except each replicate was ~ 90 females. Pooling occurred directly following quantification of newly prepared dsRNA, adding 400 ng of each dsRNA per mosquito to be injected to a microcentrifuge tube, followed by freezing aliquots at -80 °C until the day of injection. A control group of females was injected for each experiment with dsRNA targeting EGFP.
6
Targeting BLA in Genetically Engineered Mice
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Stk11f/f, Fosf/f and Fos-tTA mice were anesthetized with KXA. The skull was exposed, cleaned, and bilateral craniotomies were made at stereotactic coordinates (AP = −1.4, ML=±3.4). BLA were injected bilaterally with AAV2/5-Camk2α::Cre-GFP or AAV2/5-Camk2α::GFP (UNC, vector core) for Stk11f/f and Fosf/f mice, Camk2α::hM3Dq-mCherry or AAV2/5-Camk2α::GFP (UNC, vector core) for Stk11f/f mice and AAV2/5-TRE::mCherry for Fos-tTa mice, 10 days prior to CTA training using sterile glass micropipettes (10–20 µm diameter) attached to a partially automated microinjection device (Nanoject III Microinjector, Drummond Scientific). The micropipettes were lowered to 4.3 mm and 4.6 mm from the dura to reach the BLA. At each depth, virus (200 nl) was delivered via 10 pulses of 20 delivered every 10 s, with 10 min between each injection. Postsurgical treatment and recovery were as above.
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Stk11f/f, Fosf/f and Fos-tTA mice were anesthetized with KXA. The skull was exposed, cleaned, and bilateral craniotomies were made at stereotactic coordinates (AP = −1.4, ML=±3.4). BLA were injected bilaterally with AAV2/5-Camk2α::Cre-GFP or AAV2/5-Camk2α::GFP (UNC, vector core) for Stk11f/f and Fosf/f mice, Camk2α::hM3Dq-mCherry or AAV2/5-Camk2α::GFP (UNC, vector core) for Stk11f/f mice and AAV2/5-TRE::mCherry for Fos-tTa mice, 10 days prior to CTA training using sterile glass micropipettes (10–20 µm diameter) attached to a partially automated microinjection device (Nanoject III Microinjector, Drummond Scientific). The micropipettes were lowered to 4.3 mm and 4.6 mm from the dura to reach the BLA. At each depth, virus (200 nl) was delivered via 10 pulses of 20 delivered every 10 s, with 10 min between each injection. Postsurgical treatment and recovery were as above.
7
Insecticidal Compounds: Protocols for Injection
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Destruxin A (MedChemExpress) was resuspended in high-quality-grade dimethyl sulfoxide (DMSO) and was diluted in PBS to a 8-mM concentration. 4.6 nL of the solution or of control DMSO diluted in PBS at the same concentration was injected into flies using the Nanoject III microinjector (Drummond). Restrictocin (Sigma) was resuspended in PBS to the concentration of 1 mg/mL, 4.6 nL was injected. Beauvericin (Sigma) was resuspended in high-quality-grade DMSO. 20 mM, 9.2 nL was injected.
8
Optogenetic Manipulation of VLS Neurons
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Mice were placed in a stereotaxic frame and anesthetized with ~1–1.5% isoflurane. Small holes were drilled bilaterally at AP: +0.2 mm and ML: + / - 2.5 mm relative to bregma. Stereotaxically guided injections of (200 nL per hemisphere) were made at AP: +0.2 and ML: 2.5 and DV: −4.6 and −4.4 relative to bregma using a Nanoject III microinjector (Drummond Scientific, Broomall, PA). 100 µm-core optic fibers (Precision Fiber Products, Chula Vista, CA) were implanted with the same AP and ML coordinates and at DV: −4.2 in order to target cell bodies of the VLS. Fibers were secured to the skull with screws and dental cement along with a head post for head fixation.
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