Prior to dissection, mice were euthanized with 10% Nembutal and subsequent cervical dislocation. The gastrocnemius muscle was dissected, snap-frozen in liquid-nitrogen-cooled isopentane and stored at −80°C. On the day of the experiment, longitudinal cryosections of 20 μm were fixed in 4% PFA for 10 min before rinsing with 1X PBS. Tissue was subsequently permeabilized with PBS-T for 5 min and blocked in PBS-T supplemented with 10% normal donkey serum (NDS, Sigma-Aldrich) for 1 h. Neuronal axons (anti-NFL light chain antibody (Alexa Fluor 488 conjugated, Cell Signaling, 8024S, 1/500), presynaptic terminals (anti-SV2A, Developmental Studies Hybridoma Bank, 1/100), and motor endplates (Alexa Fluor 555-conjugated α-bungarotoxin, Life Technologies, B35451, 1/5,000) were stained with primary antibodies dissolved in PBS-T supplemented with 10% NDS and incubated overnight. Slides were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) and images were acquired with a Leica TCS SP8 confocal laser scanning microscope (Leica Microsystems). For each experimental condition, at least 100 NMJs were analyzed for innervation on a Zeiss Imager M1 microscope system using a 40X objective (Carl Zeiss, AG). Assessments were undertaken by an investigator blinded to the different groups.
Alexa fluor 555 conjugated α bungarotoxin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa-Fluor 555-conjugated α-bungarotoxin is a fluorescently labeled compound used for the detection and quantification of nicotinic acetylcholine receptors (nAChRs) in biological samples. It binds specifically to nAChRs with high affinity.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
Normal donkey serum (nds), by Merck Group (1 mentions)
Anti sv2a, by Developmental Studies Hybridoma Bank (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Tcs sp8 confocal laser scanning microscope, by Leica (1 mentions)
Hrp conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti rabbit igg ap307p, by Merck Group (1 mentions)
Hrp conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Anti app, by Abcam (1 mentions)
C myc antibody, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Synaptic Systems (1 mentions)
Anti bace1 d10e5, by Cell Signaling Technology (1 mentions)
Anti flag, by Merck Group (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
D tubocurarine, by Bio-Techne (1 mentions)
Plan apochromat 63 1.40 objective, by Zeiss (1 mentions)
Anti vacht, by Synaptic Systems (1 mentions)
Confocal microscope, by Olympus (1 mentions)
Tissue tek oct, by Sakura Finetek (1 mentions)
8 protocols using alexa fluor 555 conjugated α bungarotoxin
1
Quantitative Analysis of Neuromuscular Junction Innervation
2
Antibody-based Protein Detection Protocol
3
Quantifying Nicotinic Receptor Expression in GBM Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GBM cells were grown on laminin-covered glass placed on 24-well plates under the same conditions described in Section 2.4 . To assess the cell expression of α7, α9, and muscle-type nAChRs, the cells were fixed with 4% PFA and then stained with Alexa-Fluor 555-conjugated α-bungarotoxin (50 nM, α-Bgt-Alexa 555, Thermo Fisher Scientific) overnight at 37 °C. The cells were washed extensively with extracellular buffer (140 mM NaCl, 2 mM CaCl2, 2.8 mM KCl, 4 mM MgCl2, 20 mM HEPES, 10 mM glucose, pH 7.4) to remove any unbound α-bungarotoxin. Controls were run simultaneously with 1 μM of unlabeled d-tubocurarine (Tocris). Twelve-bit digital images were obtained using a DuoScanMeta LSM510 laser scanning microscope (Carl Zeiss, Weimar, Germany) equipped with a Plan-Apochromat 63×/1.40 objective (numerical aperture). Image acquisition parameters were as follows: for green fluorescence—excitation at 488 nm and emission at 505–550 nm, for red fluorescence—excitation at 561 nm and emission at 575 nm. Pictures were processed with open-source ImageJ Fiji software version 1.54f.
4
Immunofluorescence and Lipid Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen tissues were sliced into 5 μm thick sections and permeabilized using a buffer containing 1.5% normal serum with 0.2% triton X-100 in TBST. After incubation with antibodies, Alexa Fluor 555-conjugated α-bungarotoxin (Thermo Fisher Scientific, Waltham, MA, USA), and nuclear staining dye, the sections were mounted with an antifade medium and visualized under a fluorescence microscope.
The sections were also stained using Oil Red O staining after fixation using formalin. In brief, after they were rinsed with 60% isopropanol, the slices were incubated with Oil Red O working solution and hematoxylin. After a wash with distilled water, the slices were mounted and visualized under a light microscope.
The immunofluorescence and Oil Red O staining intensities were quantified using Image J (NIH, Bethesda, MD, USA).
The sections were also stained using Oil Red O staining after fixation using formalin. In brief, after they were rinsed with 60% isopropanol, the slices were incubated with Oil Red O working solution and hematoxylin. After a wash with distilled water, the slices were mounted and visualized under a light microscope.
The immunofluorescence and Oil Red O staining intensities were quantified using Image J (NIH, Bethesda, MD, USA).
5
Neuromuscular junction visualization
6
Immunolabeling of Neuromuscular Junctions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Longitudinal sections (50 µm) were cut through the triceps surae, blocked for 1 h in 2% BSA in PBS with 0.2% Triton X-100, incubated overnight in primary antibodies: mouse anti-NEFM (1:250, 2H3-c, DSHB) and mouse anti-SV2A (1:250, SV2-c, DSHB). This was followed by three 2 h washes in PBS with 0.5% Triton X-100 and 2h incubation with AlexaFluor488-conjugated goat anti-mouse secondary antibody (1:500, 715-545-150 from Jackson ImmunoResearch) and AlexaFluor555-conjugated α-bungarotoxin (1:1000, B35451 from ThermoFisher) to label post-synaptic acetylcholine receptors. NMJs were imaged using a Leica Stellaris 8 WLL confocal microscope.
7
Myoblast Differentiation and AChR Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
C2C12 mouse skeletal myoblasts from ATCC (ATCC-LGC Standards S.r.l., Italy) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose (Sigma, St. Louis, MO, USA) with 10% Fetal Bovine Serum (FBS; Sigma), containing 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (all purchased from Invitrogen-Gibco, Milan, Italy). The cells were cultured for 2–3 days on 8 well chamber slides in the previous medium, then replaced with DMEM and 3% FBS, to obtain myotubes. Then the cells were incubated with Fc-nLG3, ActR-Fc, ActR-Fc-nLG3 or ActR-Fc-LG3 at 10 μM for 24h and fixed with 2% paraformaldehyde for 20 min at RT. In addition, PBS was used as negative control. The samples were stained for the Acetylcholine receptor (AChR) by incubating the cells with Alexafluor 555-conjugated α-Bungarotoxin (1:500; Invitrogen, Italy) at RT for 1 h. The cells were rinsed and coverslips were mounted with a drop of PB 0.1M. The samples were then observed with a Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems) and representative images acquired.
8
Immunofluorescence Staining of Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed in 4 % paraformaldehyde for 15–25 min at room temperature. After blocking in blocking buffer (PBS containing 5 FBS and 0.3 % Triton X-100), the cells were incubated with primary antibodies overnight at 4 °C. The cells were then rinsed with PBS three times and incubated with Alexa 488-, Alexa 555-, or Alexa 647-conjugated secondary antibodies (Thermo) for 1 h at room temperature. Nuclei were stained with 10 μg/ml Hoechst 33258 (Sigma-Aldrich, USA). The cells were then rinsed with PBS three times, mounted on slides and examined using a universal fluorescence microscope (Axiophoto, Carl Zeiss, Germany) or confocal laser-scanning microscope (LSM700, Carl Zeiss, Germany). The primary antibodies used in these analyses were as follows: HB9 (mouse IgG1, 1:200, Developmental Studies Hybridoma Bank [DSHB], USA), ISL-1 (mouse IgG2a, 1:200, DSHB, USA), βIII-Tubulin (mouse IgG2b, 1:4000, Sigma-Aldrich, USA or rabbit IgG, 1:4000, Biolegend, USA), ChAT (goat IgG, 1:200, Millipore, USA), GFP (rabbit IgG, 1:200, MBL, USA), and MyHC (mouse IgG2b, 1:200, DSHB, USA). For the observation of n-AChR clusterization, Alexa fluor 555-conjugated α-Bungarotoxin, (1 μg/mL, Invitrogen, USA) was added to samples and incubated for 1 h.
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!