We set the head angle formed by the flat part of the skull above the beak and the table to 35° and drilled a window into the skull above Area X. Area X was localized based on stereotaxic coordinates and identified through the presence of tonically firing neurons, recorded with a 0.6–1.7 MΩ tungsten electrode attached to a vertical manipulator. In each hemisphere we injected 1 μl of ibotenic acid (Tocris) near the center of Area X. Injection sites were located on average 1.5–1.9 mm medial-lateral (ML), 5.5–6.0 mm anterior-posterior (AP), and 2.8–3.5 mm dorsal-ventral (DV) from the bifurcation of the midsagittal sinus (lambda). Injections were performed using a borosilicate glass pipette (BF-120-69-10, Sutter instrument) pulled with a Picospritzer (Parker Inc.) and broken with forceps to a tip diameter of about 10 μm.
Bf120 69 10
Manufactured by Sutter Instruments
Sourced in United States
The BF120-69-10 is a precision instrument used for manipulating and controlling the environment in a laboratory setting. It features advanced temperature control capabilities and is designed to maintain consistent environmental conditions for sensitive experiments or processes.
Automatically generated - may contain errors
Lab products found in correlation
Hdg9623, by Baxter (2 mentions)
Smarter ultra low rna kit for sequencing, by Takara Bio (2 mentions)
Ibotenic acid, by Bio-Techne (1 mentions)
F7252, by Merck Group (1 mentions)
Nebnext ultra 2 dna library prep kit, by New England Biolabs (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Mp 225, by Sutter Instruments (1 mentions)
Fast green, by Merck Group (1 mentions)
Sys pv820, by World Precision Instruments (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Dam 50, by World Precision Instruments (1 mentions)
Power 1401, by Cambridge Electronic Design (1 mentions)
9 protocols using bf120 69 10
1
Ibotenic Acid Injection into Area X
2
In utero Electroporation of Murine Embryos
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Wild‐type C57BL/6J pregnant mice carrying E13.5 embryos were anesthetized using initially 4% isoflurane (Baxter, HDG9623), followed by 2–3% isoflurane during the in utero electroporation (IUE) procedure. The animals were injected subcutaneously with the analgesic (0.1 ml of metamizol, 200 mg/kg). The peritoneal cavity was surgically opened and the uterus exposed. Using borosilicate microcapillary (Sutter instruments, BF120‐69‐10), the embryos were injected intraventricularly with a solution containing 0.1% Fast green (Sigma‐Aldrich, F7252) in sterile PBS, 2 µg/µl of pSuper plasmid (either 2 µg/µl of pSuper‐shcon or 1 µg/µl of pSuper‐shCcny and 1 µg/µl of pSuper‐shCcnyl1), 0.4 µg/µl of pCAGGS GFP. The electroporations (six 50‐msec pulses of 28V at 1 s intervals) were performed using a 3‐mm diameter electrode (BTX genetronics Inc., 45‐0052INT). After surgery, mice received Metamizol in drinking water (1.33 mg/ml).
Pregnant mice were sacrificed by cervical dislocation at the indicated time points (E15.5‐E17.5), and embryonic brains were dissected, fixed in 4% PFA, overnight at 4°C and processed for cryo‐sectioning.
Pregnant mice were sacrificed by cervical dislocation at the indicated time points (E15.5‐E17.5), and embryonic brains were dissected, fixed in 4% PFA, overnight at 4°C and processed for cryo‐sectioning.
3
Single-Cell RNA Sequencing of Drosophila DH44+ Neurons
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As described previously,37 (link) individual DH44+ cells (visualized by GFP expression) were harvested from dissected fly brain under a fluorescence microscope with a glass micropipette pulled from thick-walled borosilicate capillaries (BF120-69-10, Sutter Instruments). Individual cells were immediately transferred to lysate buffer (0.9× PCR Reaction Buffer II, 1.35 mM MgCl2, 0.45% NP40, 4.5 mM DTT, 0.18 U/µL SUPERase-In, 0.36 U/µL RNase inhibitor, 12.5 nM UP1 primer, 0.045 mM dNTP mix) and underwent reverse transcription and cDNA amplification (SMARTer Ultra Low RNA Kit for Sequencing, Clonetech).
To build the cDNA library, library preparation kit (NEBNext Ultra II DNA Library Prep Kit, NEB) was used. The amplified cDNAs gained from the former step were sonicated to ~250 bp fragments by the Covaris S2 system, and fragemented cDNAs were then subjected to end-preparation, adaptor ligation, adaptor-ligated DNA cleanup and PCR amplification successively according to the supplier’s protocols. The cDNA libraries were then sequenced by Illumina Hiseq 2500 platform. The sequenced raw data were first pre-processed to remove low-quality reads, adaptor sequences and amplification primer. Reads were mapped to Drosophila genome and mapped reads were selected for further analysis. FPKM (Fragments Per Kilobase Of Exon Per Million Fragments Mapped) was used to quantify gene expression.
To build the cDNA library, library preparation kit (NEBNext Ultra II DNA Library Prep Kit, NEB) was used. The amplified cDNAs gained from the former step were sonicated to ~250 bp fragments by the Covaris S2 system, and fragemented cDNAs were then subjected to end-preparation, adaptor ligation, adaptor-ligated DNA cleanup and PCR amplification successively according to the supplier’s protocols. The cDNA libraries were then sequenced by Illumina Hiseq 2500 platform. The sequenced raw data were first pre-processed to remove low-quality reads, adaptor sequences and amplification primer. Reads were mapped to Drosophila genome and mapped reads were selected for further analysis. FPKM (Fragments Per Kilobase Of Exon Per Million Fragments Mapped) was used to quantify gene expression.
4
Glucose Modulation of Feeding Behavior in Flies
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Flies were immobilized as previously described.17 (link) Approximately 50 nL of AHL buffer (108 mM NaCl, 8.2 mM MgCl2, 4 mM NaHCO3, 1 mM NaH2PO4, 2 mM CaCl2, 5 mM KCl, 5 mM HEPES)20 (link) with or without the addition of 100 mM D-glucose was injected into the thorax of these flies with a glass micropipette. The glass micropipette was pulled from thick-walled borosilicate capillaries (Sutter Instruments, BF120-69-10) with a micromanipulator (Sutter Instruments, MP225) to control its movements. Flies’ food consumption was measured immediately after AHL injection by the MAFE assay.
5
In Utero Electroporation of Murine Embryos
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Tamoxifen-treated pregnant mice carrying E13.5 embryos were anesthetized using initially 5% isoflurane (Baxter, HDG9623), followed by 2%–3% isoflurane during the IUE procedure. Endotoxin-free plasmids (Control, pCAGGS–LoxP–Gap43-GFP–LoxP–IRES–nRFP; CA-YAP, pCAGGS–LoxP–Gap43-GFP–LoxP–CA-YAP–IRES–nRFP) were mixed on the day of surgery with Fast Green (Sigma, 0.25% final concentration) to a final plasmid concentration of 2 μg/μl in 1x PBS. Using a borosilicate microcapillary (Sutter instruments, BF120-69-10) the DNA/Fast Green mixture was intraventricularly injected, which was followed by six 50-msec pulses of 30 V at 1 s intervals (BTX genetronics Inc., 45-0052INT), using a 3-mm diameter electrode (BTX genetronics Inc., 45-0487). After the IUE, the uterus was placed back into the abdominal cavity, and the peritoneum was sutured (VICRYL 5-0, V493H). Abdominal skin was closed with clips and animal received 100 μl of painkiller (Rimadyl 1 mg/ml). Pregnant mice were sacrificed by cervical dislocation at the indicated time points (E14.5-E17.5), and embryonic brains were dissected and fixed in 4% PFA, overnight at 4°C.
6
CRISPR-Mediated Targeting of EREG in Cortical Organoids
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Electroporation of the RNP complexes for CRISPR/Cas9-mediated targeting of EREG was carried out 2 days after the first slicing of human cortical organoids at week 6 of organoid culture. Organoids that did not display ventricle structures after slicing were discarded. Organoids were transferred to a 6 cm ultra-low-attachment dish (Eppendorf, 30701011) containing Tyrode’s solution (Sigma, T2145). Using a glass microcapillary (Sutter Instrument, BF120-69-10), 0.2 to 0.5 µl of the RNP/plasmid (pCAG-GFP) mix at a final concentration of 24 µM RNP and 0.1 µg/µl plasmid diluted in 0.1% Fast Green Solution (in water) were injected into areas depicting ventricular morphology. Injections were carried out using a microinjector (World Precision Instruments, SYS-PV820) in a continuous setting. Up to five ventricles were injected per organoid. A total of 7-8 organoids were processed per replicate. After injection, organoids were transferred into an electroporation chamber containing Tyrode’s solution and electroporated with five pulses applied at 38 V for 50 ms each at intervals of 1 s (Harvard Bioscience, BTX ECM 830). Subsequently, the electroporated organoids were moved back into the culture medium and incubated for 7 days before fixation with 4% PFA.
7
Single-cell cDNA preparation protocol
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The method of single-cell cDNA preparation has been described previously (Tang et al., 2010 (link)). In brief, individual GFP+ cells were picked with a glass micropipette in situ (pipettes were pulled from thick-walled borosilicate capillaries (BF120-69-10, Sutter Instruments) for initial tip opening of 1–2 μm under a dissecting microscope and transferred into lysate buffer (0.9 × PCR Reaction Buffer II, 1.35 mM MgCl2, 0.45% NP40, 4.5 mM DTT, 0.18 U/µl SUPERase-In, 0.36 U/µl RNase inhibitor, 12.5 nM UP1 primer, 0.045 mM dNTP mix) immediately, followed by reverse transcription using oligo(dT) primers to generated first-strand cDNA. A poly(A) tail was then added to the 3′ end of the first-strand cDNA by terminal deoxynucleotidyl transferase. The cDNA was amplified by 29 cycles of PCR with universal oligo(dT) primers, and then tested by nested PCR with the primers listed in Supplementary file 1 .
8
Single-cell RNA-seq of Drosophila AKHR+ Neurons
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As described in our previously report (Yu et al., 2016 (link)), individual AKHR+ cells (marked by GFP expression) were picked from dissected fly brain under a fluorescence microscope using a glass micropipette pulled from thick-walled borosilicate capillaries (BF120-69-10, Sutter Instruments). Separated cells were transferred to lysate buffer immediately, followed by reverse transcription and cDNA amplification (SMARTer Ultra Low RNA Kit for Sequencing, Clonetech). The amplified cDNA underwent library preparation using the NEBNext Ultra II DNALibrar kit and subject to sequencing by Illumina Hiseq 2500 platform. The sequenced raw data were purified to remove low-quality reads, adaptor sequences and amplification primer before mapping to Drosophila genome. Only mapped reads were selected for further analysis. FPKM (Fragments Per Kilobase Of Exon Per Million Fragments Mapped) was used to quantify gene expression. The RNAseq data were deposited in GEO database under the accession code GSE129601.
9
Electrophysiological Recording of Neuronal Responses
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Electrical stimulation was delivered via a concentric metal electrode (o.d. = 300 μm; UB-9007, Unique Medical Co., Tokyo Japan) placed on the slice surface. The center (or sleeve) of the electrode was connected to the cathode (or anode) of the isolating unit (SS-203 J) and pulse generator (SEN-3301, Nihon Koden Co., Tokyo, Japan) in the constant current mode. Field potential was recorded using a pair of Krebs-solution-filled glass micropipettes (o.d. = 1.2 mm, i. d. = 0.69 mm, borosilicate glass; #BF120-69-10, Sutter Instruments Co., Novato, California, United States ) with fine tips made by pulling and cutting (o.d. = ca. 50 μm). One pipette (plus pole) was placed in the slice tissue, and another (minus pole) was submerged in the perfusing Krebs solution as the reference electrode. These pipettes were connected through inserted silver wires to a differential amplifier (DAM-50, World Precision Instruments Inc. Sarasota, Florida, United States ) set at frequency range of 1 to 1,000 Hz and gain of x1,000. The amplified signals were stored in a computer via an A/D interface and sampling software (Power 1401 and Signal ver 5, Cambridge Electronic Design Ltd. Cambridge, United Kingdom) after digitizing at 5 kHz.
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