Twenty-micrometre sections were obtained by cutting isopentane fresh-frozen tibialis anterior muscle perpendicular to the muscle axis with a cryostat at −20 °C (Leica, Nanterre, France). Acetylcholine receptors in the postsynaptic apparatus of neuromuscular junctions were labelled with rhodamine-conjugated α-bungarotoxin (Sigma–Aldrich). Gangliosides distribution was labelled with the cholera toxin sub-unit beta coupled with an Alexa488 dye (1/200, ThermoFisher). Photomicrographs were taken with a Nikon microscope and analysed with NIS Element 4.0 (Nikon).
Rhodamine conjugated α bungarotoxin
Manufactured by Merck Group
Sourced in Belgium
Rhodamine-conjugated α-bungarotoxin is a fluorescently labeled compound used in research applications. It consists of the snake venom toxin α-bungarotoxin conjugated to the rhodamine dye. This compound can be used to label and visualize acetylcholine receptors in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 488 dye, by Thermo Fisher Scientific (1 mentions)
Cryostat, by Leica (1 mentions)
Nis element 4, by Nikon (1 mentions)
Zen 2, by Zeiss (1 mentions)
M2 microscope, by Zeiss (1 mentions)
High resolution b w camera, by Hamamatsu Photonics (1 mentions)
Anti synaptophysin antibody, by Abcam (1 mentions)
3 protocols using rhodamine conjugated α bungarotoxin
1
Neuromuscular Junction Visualization
2
Mapping Neuromuscular Junction Innervation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tibialis anterior muscles were dissected into bundles and processed for immunofluorescence with a rabbit anti-synaptophysin antibody and a rabbit anti-neurofilament antibody (Eurogentec, Seraing, Belgium) followed by Alexa-conjugated donkey anti-rabbit (Jackson) and rhodamine-conjugated α-bungarotoxin (Sigma), as previously described [25 (link)]. Neuromuscular junctions (NMJs) analysis was performed by an independent age and genotype-blinded observer, directly under and AxioImager. M2 microscope (Zeiss) was equipped with a high-resolution B/W camera (Hamamatsu) and run by the ZEN 2 software (Zeiss). On average, 500 NMJs per animal were examined. NMJs were considered partially denervated when the presynaptic nerve terminal labelled with synaptophysin was partially observed from the postsynaptic region labelled with α-bungarotoxin, and denervated when the presynaptic nerve terminal was totally absent. Representative NMJs were imaged using the same microscope setting.
3
Assessing Neuromuscular Junction Integrity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tibialis anterior muscle samples were dissected into thin bundles under a binocular microscope. Bundles were collected from at least three different parts of the muscle. Acetylcholine receptors in the postsynaptic apparatus of neuromuscular junctions were labeled with rhodamine-conjugated α-bungarotoxin (Sigma–Aldrich). Immunofluorescent labeling of nerve terminals was performed with a rabbit polyclonal anti-synaptophysin antibody diluted 1/200 (Abcam, Cambridge, UK), and Alexa-conjugated goat anti-rabbit IgG diluted 1/500 (Jackson ImmunoResearch, Suffolk, UK). Muscle bundles were mounted onto slides, prior to fluorescence microscopy. Neuromuscular junctions were considered as denervated when the presynaptic nerve terminal was absent from the postsynaptic region. Integrity of the typical pretzel-shaped morphology of neuromuscular junctions was determined as previously described (63 (link)) by quantifying the number of separate postsynaptic gutters, which estimates the degree of fragmentation, and the number of gutter intersections, which estimates the degree of enlargement and complexity of the postsynaptic apparatus.
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!