Groups of 35 to 50 females were injected intrathoracically with ~ 276 nl of DENV-2 supernatant (viral load 7.0 × 106 genomic copies) at 5–7 days post-emergence, using a Drummond Nanoinjector II (Drummond Scientific, BrookMall, Oakbrook, IL, USA) and a borosilicate capillary needle made with a needle puller (model P-97 Sutter Instrument, USA). Surviving females were maintained on 10% sucrose solution for 14 DPI (survival period). Females were then anesthetized with CO2 and kept on ice for head dissection and body collection. After removing the head, individual heads and bodies were immediately frozen in dry ice and stored at − 80 °C for molecular analysis.
Nanoinjector 2
Manufactured by Drummond
Sourced in United States
The Nanoinjector II is a precision instrument designed for the controlled delivery of small volumes of liquids at the nanoliter scale. It features advanced microfluidic technology to enable accurate and repeatable injection of samples into a variety of applications.
Automatically generated - may contain errors
9 protocols using nanoinjector 2
1
Dengue Virus Infection Dynamics
2
GDNF Administration in Larval Zebrafish
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Effects of GDNF were assessed using human recombinant GDNF (Icosagen, Össu, Estonia) at a concentration of 1 ng/nl. GDNF was administered as brain injections using a Drummond capillary (inner diameter 0.53 mm x outer diameter 1.14 mm) and Drummond Nanoinjector II (Drummond Scientific, Brookmall, USA) at 4 dpf. Capillaries were heated and pulled for a sharp tip (Narishige PC-10, Narishige, Japan) and inserted midline, post-ocular and aimed at the tectal ventricle. Preliminary injections using dye were performed to verify the anatomical location and repeatability of injections. A total volume of 9.2 nl was injected, corresponding to 9.2 ng GDNF per larva. To rule out effects due to injection of liquid, sham injections with distilled water of the same volume were performed. Injections took place between 10:00 and 13:00 for GDNF and 14:00 and 17:00 for distilled water. Prior to injection, larvae were sedated in 0.01% MS-222 (Sigma-Aldrich, St. Louis, USA). After injection, larvae recovered in system water for approximately 48 h. At 6 dpf larvae were placed in the recording system described above. After the 24 h acclimation period, data recording was performed on 7 dpf for 20 h.
3
Intracerebral CNiFER Cell Implantation
4
Intracerebral CNiFER Cell Implantation
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CNiFERs were harvested without trypsin from 80 % confluent culture flasks, centrifuged, and re-suspended in ACSF for injection. For the in vivo dose response experiment, a open craniotomy was used. For all other in vivo experiments, a ‘thinned skull’ craniotomy34 (link) was used. CNiFER cells were loaded into a 40 μm inner-diameter glass pipette connected to a Nanoinjector II (Drummond) and injected into neocortex through the thinned skull ~200 μm from the cortical surface. CNiFERs were injected into adjacent sites within the following stereotaxic coordinates: +1 to +2 mm A/P; +1 to +2 mm M/L. After implantation in several adjacent sites (typically two injection sites per CNiFER variant), the craniotomy was sealed with a glass coverslip. A custom-built head-bar was attached to the skull with C&B-METABOND (Parkell, Inc.), and the preparation surrounding the imaging window was covered with dental cement (Dentsply). Mice were immunosuppressed by daily cyclosporine injection (20 μl/100 g, i.p., Belford Laboratories).
5
CNiFER Stimulation and Quantification
6
CNiFER Stimulation and Quantification
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After CNiFER implantation, the craniotomy was kept open and a glass pipette connected to a Nanoinjector II (Drummond) was positioned, using a Sutter manipulator, 100 μm away from the CNiFER implants. Imaging was performed under urethane anesthesia (1.5g/Kg, IP). The agonist, i.e., NE or DA, was mixed with Alexa 594 to verify the arrival of the agonist as well as the calculate dilution of the agonist between the pipet and the implant. A long train of pulses of agonist (2.3 nl pressure injections as 2 s pulses every 5 s until a steady state response was observed, typically after 30 s) was then applied next to the CNiFER implants. Control pulses of Alexa 594 in saline did not cause any FRET change. The dilution was calculated by measuring the average fluorescence of the Alexa 594 in the interstitial space of the implant and comparing it to the average fluorescence of the dye in the void immediately downstream from the pipette.
7
Tracing Midbrain Dopaminergic Neurons
8
Tracing Midbrain Dopaminergic Neurons
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After anesthesia, mice were placed in a stereotaxic frame. A small craniotomy was performed where CNiFERs were typically injected (+1.5 mm A/P, +1.5 mm M/L). Using a 10 μm inner-diameter glass pipette connected to a Nanoinjector II (Drummond), 200 nl of Fluorogold™ (Fluorochrome), prepared as 1 % (w/v) in 0.1 M cacodylate buffer, was injected (20 nl every minute) in the cortex 200 μm from the surface. After 7 d, the mice were transcardially perfused. Histological sections were scanned at 1 μm spatial resolution using a Nanozoomer (Hamamatsu) digital slide scanner. Using Neurolucida software (Microbrightfield™), outlines of midbrain, brainstem, and cerebellum were drawn and sections were aligned based on anatomical borders to yield three-dimensional reconstructions. Outlines of subsantia nigra, ventral tegmental area and locus coeruleus were defined by tyrosine hydroxylase labeled neurons. Cells double-labeled for tyrosine hydroxylase and Fluorogold™ were marked and counted. Co-labeling was confirmed by confocal microscopy.
9
Verification of ZIKV Infectivity and Replication
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In parallel to the bite/transmission experiments, 14 days after the ZIKV-infective blood meal, 10 randomly selected Ae. aegypti mosquitoes were killed quickly by cold exposure, SGs dissected, and macerated SGs used for intrathoracic inoculations of 20 naïve 3 to 5 day old Ae. aegypti (Nanoinjector II, Drummond Scientific Co., Broomal, USA). These mosquitoes were maintained post-inoculation on 10% glucose solution ad libitum for 14 days and processed by qPCR for ZIKV quantification to verify that ZIKV in the originally infected mosquitoes was both infectious and replication competent [23 ].
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