Abstract
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.
Highlights
The sheet embedding is helpful for locating muscles and neuromuscular junction (NMJ) boutons in Drosophila
The larval specimens were often curl during the dehydration process of preparing sample, which would seriously interfere with the positioning of the NMJ boutons and greatly increase the number of ultrathin sections necessary
It was easy and clear to position the different muscles and to remove the excess resin and irrelevant muscles as much as we can under light microscopy
Summary
Drosophila is a classic model animal, its neuromuscular junction (NMJ) is a well-known neuroscience research model, which is widely used in many fields such as neurological diseases (Ashley et al 2018; Banerjee et al 2017), neurodevelopment (Belalcazar et al 2021; Ramesh et al 2021; Titus et al 2021; Wang et al 2021), neurodegeneration (Johnson et al 2021), and neurosignal transmission. The classic protocols for TEM of Drosophila recommend positioning type I boutons with half-thin slices and ultrathin sections (McDonald et al 2012). The fixed muscular sample of Drosophila larvae is very thin and curled in the process of alcohol/acetone dehydration, which is more unfavorable for positioning muscle and NMJ boutons. The samples were subsequently treated with mixtures of propylene oxide and epoxy resin (1:1, 1 h; 1:2, 1 h) and with pure epoxy resin twice for 2 h at room temperature (Fig. 1C). Pre-embedding specimens for immunogold electron microscopy and other samples for TEM were dehydrated in an ethanol series (50% (1 h, − 20 °C), 70% (1 h, − 35 °C), 85% (1 h, − 35 °C), 95% (1 h, − 35 °C), and 100% (1 h, − 35 °C), treated with a mixture of ethanol and Lowicryl K4M resin (1:1, 1 h, − 35 °C; 1:2, 1 h, − 35 °C), and treated with Lowicryl K4M resin twice for 24 h at − 35 °C
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