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Improved analysis method of neuromuscular junction in Drosophila larvae by transmission electron microscopy

The Drosophila neuromuscular junction is an excellent model for neuroscience research. However, the distribution of neuromuscular junctions is very diffuse, and it is not easy to accurately locate during ultrathin sectioning, which seriously interferes with the ultrastructural analysis under electro...

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Autores principales: Guangming, Gan, Mei, Chen, Chenchen, Zhang, Wei, Xie, Junhua, Geng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer Singapore 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8732911/
https://www.ncbi.nlm.nih.gov/pubmed/34661863
http://dx.doi.org/10.1007/s12565-021-00635-6
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author Guangming, Gan
Mei, Chen
Chenchen, Zhang
Wei, Xie
Junhua, Geng
author_facet Guangming, Gan
Mei, Chen
Chenchen, Zhang
Wei, Xie
Junhua, Geng
author_sort Guangming, Gan
collection PubMed
description The Drosophila neuromuscular junction is an excellent model for neuroscience research. However, the distribution of neuromuscular junctions is very diffuse, and it is not easy to accurately locate during ultrathin sectioning, which seriously interferes with the ultrastructural analysis under electron microscopy that only has a small field of view. Here, we reported an efficient method for acquiring the ultrastructural picture of neuromuscular junctions in Drosophila larva under electron microscopy. The procedure was as follows: first, the larval sample of body wall muscle was placed between the metal mesh and was dehydrated with alcohol and infiltrated with epoxy resin to prevent the sample from curling or bending, after it was dissected and fixed into thin slices. Second, the sample was embedded in resin into a flat sheet to facilitate the positioning of the muscles. Third, carefully and gradually remove the excess resin and the cuticle of the larvae, cut off both ends of the special body segment, and trim the excess specific muscles according to the recommended ratio of trimming muscles, which would reduce the workload exponentially. At last, the trimmed sample were prepared into serial about 1000 ultrathin sections that was about total 80 microns thickness, and 30–40 sections were gathered into a grid to stain with lead citrate and uranyl acetate. This method could also be applied to the other small and thin samples such as the Drosophila embryo, ventral nerve cord and brain.
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spelling pubmed-87329112022-01-18 Improved analysis method of neuromuscular junction in Drosophila larvae by transmission electron microscopy Guangming, Gan Mei, Chen Chenchen, Zhang Wei, Xie Junhua, Geng Anat Sci Int Method Paper The Drosophila neuromuscular junction is an excellent model for neuroscience research. However, the distribution of neuromuscular junctions is very diffuse, and it is not easy to accurately locate during ultrathin sectioning, which seriously interferes with the ultrastructural analysis under electron microscopy that only has a small field of view. Here, we reported an efficient method for acquiring the ultrastructural picture of neuromuscular junctions in Drosophila larva under electron microscopy. The procedure was as follows: first, the larval sample of body wall muscle was placed between the metal mesh and was dehydrated with alcohol and infiltrated with epoxy resin to prevent the sample from curling or bending, after it was dissected and fixed into thin slices. Second, the sample was embedded in resin into a flat sheet to facilitate the positioning of the muscles. Third, carefully and gradually remove the excess resin and the cuticle of the larvae, cut off both ends of the special body segment, and trim the excess specific muscles according to the recommended ratio of trimming muscles, which would reduce the workload exponentially. At last, the trimmed sample were prepared into serial about 1000 ultrathin sections that was about total 80 microns thickness, and 30–40 sections were gathered into a grid to stain with lead citrate and uranyl acetate. This method could also be applied to the other small and thin samples such as the Drosophila embryo, ventral nerve cord and brain. Springer Singapore 2021-10-18 2022 /pmc/articles/PMC8732911/ /pubmed/34661863 http://dx.doi.org/10.1007/s12565-021-00635-6 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Method Paper
Guangming, Gan
Mei, Chen
Chenchen, Zhang
Wei, Xie
Junhua, Geng
Improved analysis method of neuromuscular junction in Drosophila larvae by transmission electron microscopy
title Improved analysis method of neuromuscular junction in Drosophila larvae by transmission electron microscopy
title_full Improved analysis method of neuromuscular junction in Drosophila larvae by transmission electron microscopy
title_fullStr Improved analysis method of neuromuscular junction in Drosophila larvae by transmission electron microscopy
title_full_unstemmed Improved analysis method of neuromuscular junction in Drosophila larvae by transmission electron microscopy
title_short Improved analysis method of neuromuscular junction in Drosophila larvae by transmission electron microscopy
title_sort improved analysis method of neuromuscular junction in drosophila larvae by transmission electron microscopy
topic Method Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8732911/
https://www.ncbi.nlm.nih.gov/pubmed/34661863
http://dx.doi.org/10.1007/s12565-021-00635-6
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