Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

Summary

This report provides a new sample preparation procedure for visualizing neuromuscular junctions in Drosophila Larvae. This method is more effective in preventing the curling of the samples compared to the traditional method and is particularly useful for Drosophila neuromuscular junction ultrastructural analysis.

Abstract

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at the Drosophila NMJ enables researchers to acquire synaptic information, encompassing both quantitative data on synapse abundance and detailed insights into their morphology. However, the diffuse distribution and limited visual range of the TEM present challenges for the ultrastructural analysis. This study introduces an innovative and efficient sample preparation method that surpasses the conventional approach. The procedure begins by placing a metal mesh at the base of a flat-bottomed bottle or test tube, followed by positioning fixed larvae samples onto the mesh. An additional mesh is placed over the samples, ensuring that they are positioned between the two meshes. The fixed samples are thoroughly dehydrated and infiltrated before proceeding with the embedding procedure. Then embedding of the samples in epoxy resin is performed in a flat sheet manner, which allows for the preparation of muscles for positioning and sectioning. Benefiting from these steps, all the muscles of Drosophila larvae can be visualized under light microscopy, therey facilitating subsequent positioning and sectioning. Excess resin is removed after locating the 6^th and 7^th muscles of body segments A₂ and A₃. Serial ultra-thin sectioning of the 6^th or 7^th muscle is performed.

Introduction

Electron microscopy is one of the most ideal methods for studying the ultrastructure of biological materials that can visually and accurately demonstrate the internal structure of cells at the nanoscale level¹. However, due to the complexity of the sample preparation process and the high cost, electron microscopy is not as popular as light microscopy. Recent advancements in electron microscopy techniques have led to significant improvements in image quality, coinciding with a remarkable reduction in the associated workload. Consequently, electron microscopy has assumed an important role in advancing scientific knowledge in diverse fields

Protocol

NOTE: The transmission electron microscopy sample preparation method used in this article has been reported previously¹⁶. It is important to note that the selection of reagents and the adjustment of dosage are necessary depending on the sample. There are many toxic chemical reagents used in the sample preparation process, therefore, the operator needs to take certain protective measures, such as wearing protective clothing and gloves and operating in a fume hood.

1. Dissection, fixation and placement procedure

1. Dissection
   1. Dissect the wandering late-3rd-instar larvae in Jan solu....

Results

The Drosophila larva body wall is composed of 30 identifiable muscle fibers arranged in a regular pattern and looks like a thin slice after dissection and fixation²¹(Figure 1A). The sample remains flat during the dehydration process due to the presence of the metal meshes (Figure 1B, C). The larval body muscle is buried in a thin plate made of epoxy resin (Figure 1D-.......

Discussion

Drosophila larva samples tend to curl up during dehydration, as the samples are thin, making it difficult to accurately locate the neuromuscular junction, thereby increasing the difficulty and workload for the sample preparation. The traditional improvement is to shorten the sample⁷, but the samples were still curled to different degrees.

In our method, there are two critical steps: first, the samples remain flat throughout the dehydration process due to the re.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1,2-Epoxypropane	SHANGHAI LING FENG CHEMICAL REAGENT CO., LTD	JYJ 037-2015	Penetrating Agent
Drosophila Stocks	Bloomington	none	The wild-type control Drosophila strains used in this research were all W1118, and were reared according to standard culture methods
Flat-bottomed glass test tubes	Haimen Chenxing Experimental equipment Company	none	Flat-bottomed glass test tubes(bottle)with sponge plug(or bottle stopper)
K4M cross-linker	Agar Scientific	Cat# 1924B	The embedding resins are based on a highly cross-linked acrylate and methacrylate formula
K4M resin (monomer B)	Agar Scientific	Lot# 631557	Resin Monomer
Polyvinyl film	Haimen Chenxing Experimental equipment Company	none	Transparent polyethylene film is the best , thickness of about 0.2mm
SPI Chem DDSA	SPI	SpI#02827-AF	Dodecenyl Succinic Anhydride
SPI-Chem DMP-30 Epoxy	SPI	02823-DA	Accelerator
SPI-Chem NMA	SPI	SpI#02828-AF	Hardner for Epoxy
SPI-PON 812 Epoxy	SPI	SPI#0259-AB	Resin Monomer
Steel mesh	Yuhuiyuan Gardening Store(online)	none	Copper or stainless steel net
Transmission electron microscopy	Hitachi H-7650	11416692	All grids (on which samples were gathered) were stained with lead citrate and observed under a transmission electron microscopy.
Ultrathin microtome	Leica UC7 ultrathin microtome	595915	All sectioning operations are carried out on a Leica UC7 ultrathin microtome using a diamond knife