Cells were fixed with 3.7% formaldehyde (Fisher Scientific, Houston, TX) for 15 minutes at room temperature, washed with PBS and incubated in 0.1 M glycine/PBS for 1 hour. Cells were then incubated overnight at room temperature in cocktails of primary antibodies as described in Table 1 . Cultures were washed thoroughly in PBS and incubated for 1 hour in a corresponding cocktail of secondary antibodies as described in Table 2 . Antibodies were applied in the presence of 10% blocking solution and 0.3% TritonX-100 in PBS. For experiments requiring only surface antigen labeling, 0.3% TritonX-100 was omitted from the labeling solution. Some cells were incubated in a PBS solution containing tetramethylrhodamine conjugated α-bungarotoxin (α-BTX, 1∶500 Invitrogen) for 1 hour at room temperature to label AChRs. Cultures were rinsed in PBS and mounted in 50% glycerol/PBS mixture containing 0.03 mg/ml ρ-phenylenediamine.
α btx
Manufactured by Thermo Fisher Scientific
Sourced in United States
The α-BTX is a laboratory instrument designed for the detection and quantification of alpha-bungarotoxin, a neurotoxin commonly used in the study of nicotinic acetylcholine receptors. The core function of the α-BTX is to provide accurate and reliable measurements of this specific molecule in research samples.
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Lab products found in correlation
α btx, by Cell Signaling Technology (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Triton x 100, by Carlo Erba (2 mentions)
Gamma counter, by PerkinElmer (2 mentions)
Formaldehyde, by Thermo Fisher Scientific (1 mentions)
Anti neurofilament, by Abcam (1 mentions)
A21203, by Thermo Fisher Scientific (1 mentions)
A21207, by Thermo Fisher Scientific (1 mentions)
Mouse anti neurofilament 200, by Merck Group (1 mentions)
Rabbit anti synaptophysin, by Thermo Fisher Scientific (1 mentions)
Reep1 antibody, by Merck Group (1 mentions)
Neun antibody, by Abcam (1 mentions)
Mbf microscope, by Nikon (1 mentions)
Ab7199, by Abcam (1 mentions)
α ltx, by Alomone (1 mentions)
Smi81, by Fortrea (1 mentions)
Alexa fluor 555 c2 maleimide, by Thermo Fisher Scientific (1 mentions)
Dsk 500 dual head stereo microscope, by Motic (1 mentions)
Moticam 1080 hdmi digital camera, by Motic (1 mentions)
Lsm780 laser scanning microscope, by Zeiss (1 mentions)
21 protocols using α btx
1
Immunofluorescence Labeling of Cultured Cells
2
Imaging Neuromuscular Junctions in Mice
3
Immunolabeling of Neuromuscular Junctions
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Split segments of the gastrocnemius muscle were permeabilized in −20°C methanol for 10 min and blocked for 1 h in PBS containing 0.5% Triton and 1% BSA. Muscle segments were subsequently incubated overnight at 4°C in a cocktail of primary antibodies diluted in blocking solution. Axons and nerve terminals were labeled with mouse anti-neurofilament 200 (Sigma. N0142, 1:500) and rabbit anti-synaptophysin (ThermoFisher. MA5-16402, 1:100) antibodies. After several rinses with PBS, muscle segments were then incubated overnight at 4°C with Alexa Fluor® 594-labeled donkey anti-mouse (invitrogen. A21203, 1:200) and anti-rabbit (invitrogen. A21207, 1:200) IgGs, and Alexa Fluor® 488 labeled α-Bungarotoxin (α-BTX, invitrogen. B13422, 1:100). Muscle segments were thoroughly rinsed with PBS and mounted on glass slides. Immunohistochemically labeled NMJs were imaged using a laser scanning confocal microscope (Zeiss LSM 710).
4
Multiprotein Immunofluorescence in Neurons
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The α-BTX (tetramethylrhodamine conjugate) antibody (Invitrogen, T1175), synaptophysin antibody (Santa Cruz Biotechnology, sc-17750), Bip antibody (CST, 3177), choline acetyltransferase (Abcam, ab18736), REEP1 antibody (Sigma-Aldrich, SAB2101976), and NeuN antibody (Abcam, ab177487) were used for immunofluorescence. The REEP1 antibody (Sigma-Aldrich, SAB2101976) was also used for western blotting.
5
Neuromuscular Junction Formation Assay
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Primary myoblasts were isolated from skeletal muscles of P0.5 mouse pups and differentiated into myotubes as previously described [10 (link)]. Myotubes were resuspended in neuronal culture medium and then plated onto coverslips with co-cultured astrocytes and hiMNs at 40 to 50 dpi. After another 4 to 7 days, these sandwich cultures of myotubes, hiMNs, and astrocytes were live-stained with rhodamine-conjugated α-BTX (Invitrogen, 1:10,000) for 1 h at 37 °C. α-BTX labeled cells were then processed for immunostaining with antibodies of SYN1 (Cell Signaling Technology, 1:500) and MHC (Sigma, 1:1000). NMJ formation frequency was presented as the percentage of NMJs on myotubes associated with hiMNs networks.
6
Quantifying Motor Endplate Morphology
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Fixed ipsilateral and contralateral biceps (from 12-week time point rats) were sectioned longitudinally from the ventromedial to superficial side, at a thickness of 14 μm. Every 4th section was collected and 20 slides for each muscle were harvested to stain with α-Bungarotoxin (α-BTX, Invitrogen, Alexa Fluor 594 conjugated, 1:500) for 30 minutes. Numbers of motor endplates (MEP) on each section were counted. 10–15 MEPs from each section and 150–200 MEPs from one muscle were randomly chosen to take photos (MBF Nikon Microscope). MEP area was measured using image J. Area range was divided into six categories: from 0 to 500 μm2 (separated by hundreds) and over 500 μm2. For every biceps, numbers of MEPs falling into each category were counted and the proportion to the total number calculated. Data was expressed as mean ± SEM. One-way ANOVA was used for MEP number assessment, while student’s t test was applied for MEP area distribution analysis.
7
Formation of Neuromuscular Junctions in vitro
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Primary myoblasts were isolated from skeletal muscles of P0.5 mouse pups and differentiated into myotubes as previously described (10 (link)). Myotubes were resuspended in neuronal culture medium and then plated onto coverslips with co-cultured astrocytes and hiMNs at 40 to 50 dpi. After another 4 to 7 days, these sandwich cultures of myotubes, hiMNs, and astrocytes were live-stained with rhodamine-conjugated α-BTX (Invitrogen, 1:10,000) for 1 hour at 37 . α-BTX labelled cells were then processed for immunostaining with antibodies of SYN1 (Cell Signaling Technology, 1:500) and MHC (Sigma, 1:1,000). NMJ formation frequency was presented as the percentage of NMJs on myotubes associated with hiMNs networks.
8
Immunohistochemical Visualization of Neuromuscular Junctions
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Sagittal sections (12 µm) or frontal sections (16 µm) were incubated with rabbit anti-neurofilament 200 (NF-H, Sigma-Aldrich; 1:500) O/N at 4°C. An Alexa Fluor 488-labeled secondary antibody was used for detection. Acetylcholine receptor (AChR) clusters (axonal projection sites) localized at postsynaptic sites were identified using Alexa Fluor 594-labeled α-bungarotoxin (α-BTX, Invitrogen; 1:500).
9
Immunostaining of Synaptic Proteins
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The following primary antibodies were employed: anti‐SNAP25 (mouse monoclonal, SMI81, mouse monoclonal, Covance, catalogue 836301, 1:200), anti‐VAMP1 (rabbit polyclonal, 1:200, Rossetto et al, 1996 ), anti‐CXCL12α (mouse monoclonal, R&D, catalogue mab350, 1:50), anti‐β3‐tubulin (rabbit polyclonal, Synaptic System, catalogue SYSY 302302, 1:200), anti‐CXCR4 (rabbit polyclonal, Abcam, catalogue ab7199, 1:50–500), anti‐GAP43 (rabbit monoclonal, Abcam, catalogue ab75810, 1:200), anti‐neurofilaments (NF) 200 (mouse monoclonal, Sigma, catalogue N0142, 1:200), α‐bungarotoxin (α‐BTx, Life Technologies, catalogue B35451, 1:200). Secondary antibodies Alexa‐conjugated (1:200) were from Life Technologies.
Purified α‐LTx was purchased by Alomone (catalogue LSP‐130). The purity of the toxin was checked by SDS–PAGE and its neurotoxicity by ex vivo mouse nerve‐hemidiaphragm preparations as previously described (Rigoni et al,2005 ). Unless otherwise stated, all reagents were purchased from Sigma.
Purified α‐LTx was purchased by Alomone (catalogue LSP‐130). The purity of the toxin was checked by SDS–PAGE and its neurotoxicity by ex vivo mouse nerve‐hemidiaphragm preparations as previously described (Rigoni et al,
10
Wholemount Muscle Dissection and Imaging
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All of the equipment required for wholemount muscle dissection have been described previously (Sleigh et al., 2014a (link)). These or similar tools can also be used to prepare the ETA for in vivo imaging, while a description of how to induce anaesthesia and perform intramuscular injections has been detailed elsewhere (Sleigh et al., 2020b (link); Turney et al., 2012 (link)). Information on the primary and secondary antibodies used for immunofluorescence are provided in Tables 1 and 2 , respectively. AlexaFluor 488 and 555 α‐bungarotoxin (α‐BTX, Life Technologies, B13422 and B35451/RRID:AB_2617152, respectively, 1:1000) were used to identify postsynaptic acetylcholine receptors (AChRs). The binding fragment of tetanus neurotoxin (HCT) was produced and labelled with AlexaFluor 555 C2 maleimide (Life Technologies, A20346) as previously described (Gibbs et al., 2016 (link)). All dissection images and videos were taken using a DSK 500 dual head stereo microscope (Motic, Barcelona, Spain, PM5539B901) with attached Moticam 1080 HDMI digital camera (Motic, MC1080). Fixed and live immunofluorescent images were taken on an inverted LSM780 laser scanning microscope (Zeiss) using a 20×, 40× or 63× objective.
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