To quantitatively compare LTP and lipophorin levels in hemolymph, we placed two washed and dry larvae on a piece of parafilm on ice and pierced them with a pair of forceps. Hemolymph was collected by capillarity filling 0.5 μl glass micropipettes (Drummond) and immediately transferring the contents to 20 μl Laemmli buffer for western blot analysis.
Glass micropipette
Manufactured by Drummond
Sourced in United States
A glass micropipette is a laboratory instrument used for precise and accurate measurement and transfer of small volumes of liquids, typically in the range of microliters. It consists of a thin, calibrated glass tube with a narrow tip, allowing for the handling of minute quantities of fluids.
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Lab products found in correlation
Fast green, by Merck Group (5 mentions)
Cunningham adaptor, by Harvard Apparatus (5 mentions)
Stereotaxic frame, by Kopf Instruments (3 mentions)
P 97 glass puller, by Sutter Instruments (3 mentions)
Stereotaxic frame, by World Precision Instruments (2 mentions)
Vertical puller, by Narishige (2 mentions)
Smiths medical pm, by Smith & Nephew (2 mentions)
P 97 puller, by Drummond (2 mentions)
Wistar rat, by Charles River Laboratories (2 mentions)
Oct compound, by Avantor (1 mentions)
Ecm 830, by Harvard Apparatus (1 mentions)
Electroporation chamber, by Harvard Apparatus (1 mentions)
Somnopentyl, by Kyoritsu Seiyaku (1 mentions)
Stearic acid, by Merck Group (1 mentions)
Bz x700, by Keyence (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Micromanipulator, by Drummond (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
25 protocols using glass micropipette
1
Quantifying LTP and Lipophorin in Larval Hemolymph
2
Electroporation of Cerebral Organoids
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COs were electroporated at two different time points: at day 20 and day 35 after plating the iPSCs on the 96-well plate. For electroporation, COs were kept in NDM+A medium without Antibiotic Antimycotic solution and moved to an electroporation chamber (Harvard Apparatus). Using a stereoscope to localize the ventricle-like cavity (VL), 1–2 μl of each plasmid (pCAGGS-GFP, pCAGGS-GFP-IRES-GNG5, 2/3 of pCAGGS-GFP + 1/3 of pCAGGS-GAP43-GFP or 2/3 of pCAGGS-GFP-IRES-GNG5 + 1/3 of pCAGGS-GAP43-GFP) to a final concentration of 1 μg/μl mixed with 0.1% Fast-Green (F7252, Sigma Aldrich) were injected using Glass Micropipettes (5-000-1001-X10, Drummond Scientific) and electroporated with five pulses applied at 80 mV for 50 ms each at intervals of 500 ms (ECM830, Harvard Apparatus). 24 hours after electroporation COs were moved to new NDM+A medium and kept in culture for 7 additional days until they were fixed for 2 h in 4% PFA. After fixation, COs were transferred to 30% sucrose in PBS overnight for cryopreservation, embedded in OCT Compound (361603E, VWR Chemicals) and stored at −20°C.
For immunohistochemistry, 14 μm sections were prepared with a cryostat. For each analysis, at least 3 different COs per condition were analyzed from 2 independent batches.
For immunohistochemistry, 14 μm sections were prepared with a cryostat. For each analysis, at least 3 different COs per condition were analyzed from 2 independent batches.
3
Stereotaxic Tracer Injections in TH-Cre Rats
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All stereotaxic injections were made using glass micropipettes (tip diameter 10‐15 µm; Drummond Scientific Company, Broomall, PA, USA). Male TH‐Cre rats (7‐9 weeks old, body weight 300 g ± 25 g) were anaesthetised with isoflurane, fixed in a stereotaxic frame, craniotomised and the pipette inserted into the target brain region with reference to bregma and lambda. Following the postoperative intervals appropriate for each tracer, rats were perfused transcardially as above and the brains removed and processed for immunofluorescence. Only rats with injection sites entirely restricted to areas of interest were analysed.
4
Stereotactic Viral Vector Delivery to Mouse Zona Incerta
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Mice were anesthetized with gas isoflurane (5% for induction and 1.5% for maintenance) in O2 (Provet/Primal Healthcare, EZ Anesthesia Systems) and placed onto the stereotaxic frame (World Precision Instruments). Lidocaine (0.2 mg/g) (Streuli Pharma) was injected subcutaneously above the skull. The skin was disinfected with 70% ethanol and Betadine (Mundipharma). A small incision along the anterior-posterior axis was made at the midline, and then, the skull was leveled. A small hole was drilled through the skull above the ZI, which consists of the regions ZI and subincertal nucleus in the mouse atlas (1.3 mm posteriorly to the bregma, ±1.3 mm laterally to the midline, and −4.87 mm deep from the surface of the skull at a 10° angle away from the midline) (54 ). Glass micropipettes (Drummond) were pulled with a vertical puller (Narishige). Viral vectors were ejected into the ZI with a pressure injector system controlled by a pulse generator (A.M.P.I.; 10 to 100 ms, 20 psi, at 0.333 Hz). The wound was either stitched together with tissue absorptive silk sutures (SABANA) or continued with additional implantations (optic fiber, cannula, or GRIN lens). Buprenorphine (Bupaq P, Streuli Pharma AG) pain killers were administered (0.1 μg/g) after surgery as needed.
5
Embryo Transfer in Mouse Pseudopregnancy
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Embryos were transferred into the oviduct of a pseudopregnant recipient using glass micropipettes (150 μm inner diameter, Drummond Scientific Company, Broomall, PA, USA) under anesthesia
with pentobarbital sodium (0.035 mg/g body weight; Somnopentyl, Kyoritsu Seiyaku Co., Ltd., Tokyo, Japan). Two to four embryos per oviduct were transferred (4-8 per individual). Owing to the
small number of embryos, recipient No. 4 had two embryos transferred to only one side of the oviducts. Pseudopregnant recipients were generated according to the following treatment
protocols: treatment 1, animals were injected with hCG (10 IU/0.1 ml/head) and mated with vasectomized males 48 h before embryo transfer; treatment 2, animals were injected with hCG and
mated with vasectomized males 24 h before embryo transfer; treatment 3, animals injected with hCG 24 h before embryo transfer. Specifically, 2- to 4-cell-stage embryos developed in
vivo were transferred to recipients receiving treatment 1, 2, or 3, whereas in vitro cultured 2- to 4-cell-stage embryos were transferred to recipients receiving
treatment 2. The delivery and number of offspring were examined at 33 and 34 days after embryo transfer.
with pentobarbital sodium (0.035 mg/g body weight; Somnopentyl, Kyoritsu Seiyaku Co., Ltd., Tokyo, Japan). Two to four embryos per oviduct were transferred (4-8 per individual). Owing to the
small number of embryos, recipient No. 4 had two embryos transferred to only one side of the oviducts. Pseudopregnant recipients were generated according to the following treatment
protocols: treatment 1, animals were injected with hCG (10 IU/0.1 ml/head) and mated with vasectomized males 48 h before embryo transfer; treatment 2, animals were injected with hCG and
mated with vasectomized males 24 h before embryo transfer; treatment 3, animals injected with hCG 24 h before embryo transfer. Specifically, 2- to 4-cell-stage embryos developed in
vivo were transferred to recipients receiving treatment 1, 2, or 3, whereas in vitro cultured 2- to 4-cell-stage embryos were transferred to recipients receiving
treatment 2. The delivery and number of offspring were examined at 33 and 34 days after embryo transfer.
6
Viral Transduction of Zona Incerta in Mice
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Mice were anesthetized with gas isoflurane (5% for induction and 1.5% for maintenance) in O2 (Provet/Primal Healthcare, EZ Anesthesia Systems) and placed onto the stereotaxic frame (World Precision Instruments). Lidocaine (0.2mg/g) (Steuli Pharma) and Betadine (Mundipharma). A small incision along the anterior-posterior axis was made at the midline and then the skull was leveled. A small hole was drilled through the skull above the ZI ((44); 1.3mm posteriorly to the Bregma, ±1.3mm laterally to the midline, and -4.87 mm deep from the surface of the skull at a 10 degrees angle away from the midline).
Glass micropipettes (Drummond) were pulled with a vertical puller (Narishige). Viral vectors were ejected into the ZI with a pressure injector system controlled by a pulse generator (A.M.P.I.; 10-100ms, 20psi, at 0.333 Hz). The wound was either stitched together with tissue absorptive silk sutures (SABANA) or continued with additional implantations (optic fiber, cannula, or gradient refractive index lens). Buprenorphine (Bupaq P, Steuli Pharama AG) pain killers were administered (0.1µg/g) post-surgery as needed.
Glass micropipettes (Drummond) were pulled with a vertical puller (Narishige). Viral vectors were ejected into the ZI with a pressure injector system controlled by a pulse generator (A.M.P.I.; 10-100ms, 20psi, at 0.333 Hz). The wound was either stitched together with tissue absorptive silk sutures (SABANA) or continued with additional implantations (optic fiber, cannula, or gradient refractive index lens). Buprenorphine (Bupaq P, Steuli Pharama AG) pain killers were administered (0.1µg/g) post-surgery as needed.
7
In-utero Electroporation for Genetic Manipulation
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Rcor2 shRNA sequences were cloned into pSicoR-RFP vector, Rcor2 sequences were cloned into pMX-FLAG-HA-IRES-GFP vector and Shh shRNA sequences were cloned into pLL3.7 vector, as previously described51 (link). In-utero electroporation was performed as previously described51 (link). In brief, a timed pregnant CD-1 mouse or Rcor2fl/fl mouse at E13.5 was anaesthetized, the uterine horns were exposed and 1 μl of plasmid DNA (1–3 μg μl−1) mixed with Fast Green (Sigma) was manually microinjected through the uterus into the lateral ventricle, using a beveled and calibrated glass micropipette (Drummond Scientific). For electroporation, five 50-ms pulses of 40–50 mV with a 950-ms interval were delivered across the uterus with two 9-mm electrode paddles positioned on either side of the head (ECM830, BTX). After the procedure, the uterus was placed back in the abdominal cavity and the wound was surgically sutured. The mouse was then placed in a 28 °C recovery incubator under close monitoring until it recovered and resumed normal activity. Related primer sequences were listed in Supplementary Table 1 .
8
Preparation and Characterization of Lipid Monolayers
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Monolayers of lipid mixtures were prepared on a computer-controlled Langmuir film balance (USI System, Fukuoka, Japan) calibrated using stearic acid (Sigma-Aldrich, St. Louis, MO). The lipid solution was prepared by dissolving appropriate amount of DSPC and DOPC in chloroform/methanol (4:1 v/v). Then, 30 μL of lipid solution (1 mg/mL) was spread onto the water subphase (100 × 290 mm2) with a glass micropipette (Drummond Scientific Company, Pennsylvania, USA). The monolayers were compressed at a rate of 20 mm2/s after an initial delay period of 10 min for the evaporation of organic solvents. The subphase and ambient temperatures were controlled at 25.0 ± 0.1 °C and 25 ± 1 °C, respectively. The measurements were repeated three times under the same conditions and average data were shown.
In the preparation of the supported monolayer, the lipid solution in the presence of 0.2 mol% Texas Red-DPPE was spread on the water subphase. Then, the substrate such as a mica plate and a collodion coated TEM-grid (Okenshoji Co., Ltd, Tokyo, Japan) was horizontally dipped into the water subphase, and the lipid sample was compressed to 30 mN/m at the rate of 20 mm2/s. After the compression, the monolayer was transferred onto the substrate. Then, distribution of the ordered and disordered phases in the supported monolayers were observed with a fluorescent microscope BZ-X700 (Keyence, Osaka, Japan).
In the preparation of the supported monolayer, the lipid solution in the presence of 0.2 mol% Texas Red-DPPE was spread on the water subphase. Then, the substrate such as a mica plate and a collodion coated TEM-grid (Okenshoji Co., Ltd, Tokyo, Japan) was horizontally dipped into the water subphase, and the lipid sample was compressed to 30 mN/m at the rate of 20 mm2/s. After the compression, the monolayer was transferred onto the substrate. Then, distribution of the ordered and disordered phases in the supported monolayers were observed with a fluorescent microscope BZ-X700 (Keyence, Osaka, Japan).
9
Stereotaxic AAV delivery of fluorescent biosensors
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PCR amplified roGFP, mitoroGFP, truncated MAO-B lacking C-terminus tail sequence required for anchoring (Amino acids 492–520), Perceval HR (Addgene plasmid #49082) 12 (link), or roGFP with the C-terminal anchoring sequence of MAO-B were subcloned into EcoRI and SalI restriction sites of the pFB-TH-SV40 vector and packaged into rAAVs using serotype 9 with titers 2.1-x1013 viral genome copies/ml (Virovek). Mice were anesthetized using an isoflurane precision vaporizer (Smiths Medical PM) and placed in a stereotaxic frame (David Kopf Instruments) with a Cunningham adaptor (Harvard Apparatus) to maintain anesthesia delivery throughout surgery. After exposing the skull, a small hole was drilled and 350 nL of viral vector delivered via a glass micropipette (Drummond Scientific Company) pulled on a Sutter P-97 puller. The substantia nigra pars compacta (SNc) was targeted at the following coordinates: AP: −3.05, ML: 1.20, and DV −4.30. All surgeries were performed in wild-type or MAO-A/B knockout mice. Experiments in animals with stereotaxic delivery of AAV viral vectors were performed after at least 10 days post-op.
10
Stereotaxic AAV delivery of fluorescent biosensors
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