Native BoNT/C and BoNT/A1 were purified as previously described [66 (link), 67 (link)]. Cytosine β-D-arabinofuranoside hydrochloride (C6645), DNAse I from bovine pancreas (DN25), poly-L-lysine hydrobromide (P1274) and trypsin (T4799) were from Sigma Aldrich. μ-Conotoxin GIIIB is from Alomone, Jerusalem, Israel. Primary antibodies: anti-SNAP-25 (SMI81, ab24737) was from Abcam. Anti-SNAP-25 (cleaved) and syntaxin-1A/1B polyclonal antibodies were produced in our laboratory and previously characterized [37 (link), 68 (link)]. Secondary antibodies conjugated to HRP were from Calbiochem; secondary antibodies for immunofluorescence conjugated to Alexa Fluorophores 488 or 555 and α-Bungarotoxin conjugated to Alexa 647 were from Thermo Scientific, Waltham, MA, USA.
μ conotoxin giiib
Manufactured by Alomone
Sourced in Israel, Palestine, State of
μ-conotoxin GIIIB is a small peptide toxin derived from the venom of the cone snail Conus geographus. It functions as a selective and potent blocker of voltage-gated sodium channels.
Automatically generated - may contain errors
Lab products found in correlation
Clampfit software, by Molecular Devices (2 mentions)
S88 stimulator, by Natus (2 mentions)
Ni pci 6221, by National Instruments (2 mentions)
Cytosine β d arabinofuranoside hydrochloride c6645, by Merck Group (1 mentions)
Poly l lysine hydrobromide p1274, by Merck Group (1 mentions)
Trypsin t4799, by Merck Group (1 mentions)
Clampex 10, by Molecular Devices (1 mentions)
Digital camera, by Nikon (1 mentions)
Monoclonal antibody labeling kit, by Thermo Fisher Scientific (1 mentions)
Upright fluorescence microscope, by Olympus (1 mentions)
Bx50wi upright microscope, by Olympus (1 mentions)
Pclamp, by Molecular Devices (1 mentions)
Silicone coated surface, by Dow (1 mentions)
Blebbistatin, by Merck Group (1 mentions)
D tubocurarine, by Merck Group (1 mentions)
14 protocols using μ conotoxin giiib
1
Purification and Characterization of BoNT/C and BoNT/A1
2
Electrophysiological Assessment of Neuromuscular Junctions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were injected in the left hind limb as described for the DAS assay with indicated doses. At scheduled times, mice were sacrificed by anesthetic overdose coupled to cervical dislocation and the soleus muscle dissected. Electrophysiological recordings were performed in oxygenated Krebs-Ringer solution, using intracellular glass microelectrodes (WPI) filled with 1 M KCl and 2 M CH3COOK. Evoked junction potentials (EJP) were recorded in current-clamp mode, starting from resting membrane potential of -70 mV, adjusted with direct current injection if needed. EJPs were elicited by supramaximal nerve stimulation at 0.5 Hz, using a suction microelectrode connected to a S88 stimulator (Grass, Warwick, RI, USA). Muscle contraction was prevented by 1 μM μ-Conotoxin GIIIB (Alomone, Jerusalem, Israel). Signals were amplified with intracellular bridge mode amplifier (BA-01X; NPI, Tamm, Germany), sampled using a digital interface (NI PCI-6221; National Instruments, Austin, TX, USA) and recorded by means of electrophysiological software (WinEDR; Strathclyde University, Glasgow, Scotland, UK). EJPs measurements were carried out with Clampfit software (Molecular Devices, Sunnyvale, CA, USA). EJPs represent the average value obtained analyzing at least three muscles (15 fibers/muscle) for each condition at each time-point and reported as a percentage with respect to control muscles.
3
Inhibition of Muscle Contraction Using μ-Conotoxin GIIIB
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To block muscle stimulation μ-conotoxin GIIIB (#C-270, Alomone Labs Ltd, Jerusalem, Israel) was used. This toxin inhibits sarcolemmal voltage-dependent sodium channels (VSDCs) without affecting synaptic ACh release or ACh signaling [42 (link)]. It was supplied as lyophilized powder of > 99% purity. μ-conotoxin GIIIB was 150 μM stock, and working concentration was 1.5 μM in Ringer’s solution [mM: NaCl 137, KCl 5, CaCl2 2, MgSO4 1, NaH2PO4 1, NaHCO3 12, glucose 12.1, and DMSO 0.1%, oxygenated with O2:CO2 (95:5)].
PKA activity was blocked with N-[2-((p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89, Calbiochem). H-89 was made as 10 mM stock and used at 10 μM diluted in Ringer’s solution with DMSO.
All chemicals were diluted in Ringer’s solution, and both control and drug-containing solutions contained 0.1% dimethyl sulfoxide (DMSO) as the vehicle.
PKA activity was blocked with N-[2-((p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89, Calbiochem). H-89 was made as 10 mM stock and used at 10 μM diluted in Ringer’s solution with DMSO.
All chemicals were diluted in Ringer’s solution, and both control and drug-containing solutions contained 0.1% dimethyl sulfoxide (DMSO) as the vehicle.
4
Neuromuscular Synaptic Transmission Recordings
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Synaptic transmission recordings were performed in acute neuromuscular preparations of the TVA muscle at room temperature. A glass microelectrode (10–20 MΩ) filled with 3 M KCl was connected to an intracellular recording amplifier (TEC-05X; npi electronic) and used to impale single muscle fibers near the motor nerve endings. Evoked EPPs and mEPPs were recorded as described previously (7 (link)). Muscle contractions were prevented by including in the bath 3–4 μM μ-conotoxin GIIIB (Alomone Laboratories). The mean amplitudes of the EPP and mEPPs recorded at each NMJ were linearly normalized to −70 mV resting membrane potential, and EPPs were corrected for non-linear summation (52 (link), 53 (link)).
5
Quantification of Neuromuscular Junction Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Experiments were performed at room temperature in a recording chamber containing 1 ml of 50% Neurobasal/50% Hibernate low fluorescence solution (Brain Bits, Springfield, IL) supplemented with B27 (Sigma). Postsynaptic endplates were identified by the application of a non-blocking AChR antibody, mAb35 (Table 1 ) conjugated to an Alexa Fluor546 fluorchrome using a Monoclonal Antibody Labeling Kit (Invitrogen) for 1 hour prior to the recording session. NMJs were identified by the expression of GFP in apposition to mAb35 fluorescence identified using a CCD camera coupled to an Olympus upright fluorescence microscope (Centre Valley, PA). Images were captured using a Nikon digital camera. Micropipettes used for recordings had tip resistances between 10 and 50 MΩ and were filled with 3 M KCl. Reponses were recorded with a Sutter amplifier and processed with Clampex 10.2 software (Molecular Devices). All data were analyzed using MiniAnalysis (Synaptosoft, Decatur, GA). Quantal contents were determined by the direct method (m = spontaneous endplate potential/miniature endplate potential; sEPP/mEPP) using the mean mEPP as determined following application of 2.5 μM TTX. In some cases, 5 μM μ-conotoxin GIIIB (Alomone Labs, Jerusalem, Israel) was added to the recording solution to block Na+ channel-mediated myotube contraction.
6
Vascular Regulation via Chemical Modulators
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isoprenaline hydrochloride (Iso), sodium nitroprusside dehydrate (SNP) and ethyl 3-aminobenzoate methanesulfonate (MS-222, tricaine) were purchased from Sigma Aldrich. Iso is a β1/β2-adrenoreceptor agonist [15 (link),25 ,26 (link)], whereas sodium nitroprusside (SNP) is a potent nitric oxide donor. Nitric oxide plays a major role in regulating the vascular diameter, endothelial cell migration and angiogenesis [27 (link),28 (link),29 (link),30 (link)]. The paralytic agent μ-conotoxin GIIIB was obtained from Alomone Labs, Israel.
7
Phrenic nerve-diaphragm preparation for electrophysiology
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The diaphragm with the phrenic nerve attached was quickly dissected from the mouse and bathed in oxygenated Ringer’s solution containing (in mM): 10 Glucose, 125 NaCl, 25 NaHCO3, 1.25 NaH2PO4.2H20, 2.5 KCl, 1.8 CaCl2, 1 MgCl2, pH 7.4 (300–310 mOsm). Under the microscope, the nerve was drawn into the suction electrode by applying gentle suction. Muscle contraction to nerve stimulation was tested by applying a single pulse of stimulation to ensure the viability of the phrenic nerve-diaphragm preparation. Stimulation was performed with the A365 stimulation isolator (WPI). Muscle contraction was subsequently blocked using the muscle sodium channel blocker, μ-conotoxin GIIIb (2 μM; Alomone, C-270). Spontaneous miniature endplate potential (mEPP) and nerve-evoked endplate potential (EPP) recordings were made at room temperature under the current clamp with multi-clamp 200B amplifier. The recording electrode solution is made up of 2 M potassium gluconate and 10 mM KCl. EPP and mEPP recordings were started 30 min after the diaphragm was placed in the bath solution. Only fibers with a stable resting membrane potential ≤ −60 mV were analyzed. On average, 8 myofibers were recorded per diaphragm.
8
Electrophysiological Recordings of Neuromuscular Junctions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recordings were performed as per our previous study [46 (link)]. In brief, fine tipped glass microelectrodes (30 to 50 MΩ) filled with 2 M KCl were used to record endplate potentials (EPPs), miniature endplate potentials (mEPPs) and resting membrane potentials (RMPs). Electrodes were manipulated to impale muscle fibers within 0.5 mm of the endplate region. Proximity to endplate regions was confirmed through analysis of EPP and mEPPs rise times, sites with rise times greater than 1.5 ms were rejected. During recordings the initial RMP values were in the range of 70-85 mV with these values undergoing a gradual decrease to steady values between 55-60 mV. Recordings were terminated if the RMP fluctuated by more than 10% from the steady values. After a site was located stimulus was ceased for a rest period of ten minutes. The sodium channel blocker, μ-Conotoxin GIIIB (0.5-2 μM, Alomone Labs) was used to prevent stimulus-induced muscle contractions at elevated [Ca2+]O.
9
Electrophysiological analysis of motor neuron disease
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The EDL muscle was dissected from control and SMN2 1-copy Smn1ΔMPC mice, along with the peroneal nerve, and then pinned to a Sylgard-coated recording chamber. Intracellular recording was conducted in oxygenated Ringer’s solution, which comprised 138.8 mM NaCl, 4 mM KCl, 12 mM NaHCO3, 1 mM KH2PO4, 1 mM MgCl2, and 2 mM CaCl2 with a pH of 7.4. Action potential of the muscle was prevented by preparing the muscle in 2.5 μM μ-conotoxin GIIIB (Alomone, Jerusalem, Israel) for 10 min beforehand. The recording was performed in toxin-free Ringer’s solution. mEPPs were recorded from a junction, followed by recordings of eEPPs by stimulating the attached peroneal nerve. The eEPPs were elicited using evoked stimulation. Paired-pulse stimulation (10-ms interstimulus interval) was utilized to assess synaptic transmission. The data were obtained and analyzed with Axoclamp 900A and Clampfit version 10.7 software.
10
Intracellular Recording of Neuromuscular Junction Potentials
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The electrical stimulation and intracellular recording were performed as previously described42 (link). Briefly, the nerve was stimulated by means of a suction electrode. The stimulation consisted of square-wave pulses at variable frequencies. A glass microelectrode filled with 3M KCl was connected to an intracellular recording amplifier (Neuro Data IR283, Cygnus technology) and used to impale single muscle fibers near the motor nerve endings. Evoked endplate potentials (EPP) and miniature EPPs (mEPPs) were recorded from different NMJs within the muscle as described previously. Muscle contraction was prevented by including in the bath 3–4 μM μ;-conotoxin GIIIB (Alomone Laboratories), a specific blocker of muscular voltage-gated sodium channels. The data were analyzed as previously described42 (link). EPP Amplitudes were normalized to −70 mV and corrected for non-linear summation.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!