Name: optimizing sample preparation process for transmission electron microscopy of neuromuscular junctions in drosophila larvae
Uploaded: 2023-09-15T14:00:00
Description: Watch this Scientific Journal Video about Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae at JoVE.com

This work was supported by Natural Science Foundation of China Grant 32070811, and Southeast University (China) Analysis Test Fund 11240090971. We thank the Laboratory of Electron Microscopy and Center of Morphological Analysis, School of Medicine, Southeast University, Nanjing, China.

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.
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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...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64934">Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae</a>. The Authors section was updated from:
Gan Guangming1,2 
Sheng Qingyuan3 
Ou Yutong1 
Chen Mei1 
Zhang Chenchen1 
1The School of Medicine, Southeast University, 
2The Key Laboratory of Developmental Genes and Human Disease, Southeast University, 
3The School of Life Science and Technology, Southeast University 
Corresponding Authors:&#160;Gan Guangming
to:
Gan Guangming1,2 
Sheng Qingyuan3 
Ou Yutong1 
Chen Mei3 
Zhang Chenchen1 
1The School of Medicine, Southeast University, 
2The Key Laboratory of Developmental Genes and Human Disease, Southeast University, 
3The School of Life Science and Technology, Southeast University 
Corresponding Authors:&#160;Gan Guangming,&#160;Sheng Qingyuan

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64934">Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae</a>. The Authors section was updated.

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

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 fields2.
Drosophila is an excellent animal model for performing genetic manipulations to precisely control the spatial and temporal expression of target genes3. Besides, Drosophila has the advantages of a short growth period and easy rearing compared to mammalian models; therefore, Drosophila is widely used in morphology research4,5.
In Drosophila larvae, neuromuscular junction (NMJ) boutons are widely distributed in the muscles6,7, and immunostaining of NMJ can easily provide information on synapse quantity and morphology8,9 . The NMJ boutons located in the 6th/7th muscles of the A2 and A3 segments are well suited for quantitative and morphological research using light microscopy. This is because of their size and abundance10,11. Therefore, Drosophila larvae NMJs are considered a useful model for neuroscience research12. 
However, it is challenging to observe the NMJ bouton ultrastructure by the TEM. Since the scan window of transmission electron microscopy is narrow, it is hard to position the widely distributed NMJ boutons13. The other reason is that the Drosophila body wall is susceptible to curling during the alcohol dehydration step of the sample preparation protocol7.
Traditional studies have usually chosen boutons between the 6th and 7th muscles of the A2 and A3 segments as sample materials because of their abundance and size14,15. The 6th and 7th muscles of the A2 and A3 segments are bigger than the other muscles and contain more boutons. However, when samples were prepared for electron microscopy, fixed samples tended to become thin and prone to curling, thereby leading to improper positioning of the 6th and 7th muscles of the A2 and A3 segments.
We hereby report a new processing procedure that is more effective in preventing the curling of the samples compared to the traditional method of sample preparation7,16, by allowing the samples to stay flat during the subsequent dehydration, thereby facilitating better positioning of the Drosophila larval neuromuscular junctions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,2-Epoxypropane</td>
			<td>SHANGHAI LING FENG CHEMICAL REAGENT CO., LTD</td>
			<td>JYJ 037-2015</td>
			<td>Penetrating Agent</td>
		</tr>
		<tr>
			<td>Drosophila Stocks</td>
			<td>Bloomington</td>
			<td>none</td>
			<td>The wild-type control Drosophila strains used in this research were all W1118, and were reared according to standard culture methods</td>
		</tr>
		<tr>
			<td>Flat-bottomed glass test tubes</td>
			<td>Haimen Chenxing Experimental equipment Company&#160;</td>
			<td>none</td>
			<td>Flat-bottomed glass test tubes(bottle)with sponge plug(or bottle stopper)</td>
		</tr>
		<tr>
			<td>K4M cross-linker</td>
			<td>&#160;Agar Scientific</td>
			<td>Cat# 1924B</td>
			<td>The embedding resins are based on a highly cross-linked acrylate and methacrylate formula&#160;</td>
		</tr>
		<tr>
			<td>K4M resin (monomer B)</td>
			<td>&#160;Agar Scientific</td>
			<td>Lot# 631557</td>
			<td>Resin Monomer</td>
		</tr>
		<tr>
			<td>Polyvinyl film</td>
			<td>Haimen Chenxing Experimental equipment Company&#160;</td>
			<td>none</td>
			<td>Transparent polyethylene film is the best , thickness of about 0.2mm</td>
		</tr>
		<tr>
			<td>SPI Chem DDSA</td>
			<td>SPI</td>
			<td>SpI#02827-AF</td>
			<td>Dodecenyl Succinic Anhydride</td>
		</tr>
		<tr>
			<td>SPI-Chem DMP-30 Epoxy</td>
			<td>SPI</td>
			<td>02823-DA</td>
			<td>Accelerator</td>
		</tr>
		<tr>
			<td>SPI-Chem NMA</td>
			<td>SPI</td>
			<td>SpI#02828-AF</td>
			<td>Hardner for Epoxy</td>
		</tr>
		<tr>
			<td>SPI-PON 812 Epoxy</td>
			<td>SPI</td>
			<td>SPI#0259-AB</td>
			<td>Resin Monomer</td>
		</tr>
		<tr>
			<td>Steel mesh&#160;</td>
			<td>Yuhuiyuan Gardening Store(online)</td>
			<td>none</td>
			<td>Copper or stainless steel net</td>
		</tr>
		<tr>
			<td>Transmission electron microscopy</td>
			<td>Hitachi H-7650</td>
			<td>11416692</td>
			<td>All grids (on which samples were gathered) were stained with lead citrate and observed under a transmission electron microscopy.</td>
		</tr>
		<tr>
			<td>Ultrathin microtome</td>
			<td>Leica UC7 ultrathin microtome</td>
			<td>595915</td>
			<td>All sectioning operations are carried out on a Leica UC7 ultrathin microtome using a diamond knife</td>
		</tr>
	</tbody>
</table>

optimizing sample preparation process for transmission electron microscopy of neuromuscular junctions in drosophila larvae

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 6th and 7th muscles of body segments A2 and A3. Serial ultra-thin sectioning of the 6th or 7th muscle is performed.

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-...

Watch this Scientific Journal Video about Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae at JoVE.com

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

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.

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

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at ...

The diffused distribution and limited visual range of the TEM present challenges for the infrastructural analysis. This protocol presents an innovative and efficient sample preparation method that surpasses the conventional approach. This advanced method of TEM sample preparation is more effective in preventing the curling of the samples compared to the traditional method by allowing the samples to stay flat during the subsequent dehydration.The selection of reagents, and adjustment of dosage must be necessary depending on the sample type. There are many toxic chemical reagents used in the sample preparation process. Demonstrating the procedure will be Sheng Qingyuan, a master student from Dr.Gan Guangming's laboratory.To begin, dissect the wandering late third instar Drosophila larvae in Jan solution, and obtain the entire body wall per the standard procedures. Fix the larval body wall with the fixative in the dissection chamber overnight at four degrees Celsius. The next day, transfer the larval body wall from the dissection chamber to a 1.5 milliliter centrifuge tube, and rinse it several times with sodium cacodylate buffer.Fix the larval body wall in 1%osmium tetroxide for two hours. After fixation, wash the samples several times with distilled water. Add 2%saturated urinal acetate solution into the tube to stain the PHI neuromuscular junction.And after two hours, rinse the samples with deionized water. Next using scissors, cut two round pieces of metal mesh of pore size 270 microns. Place the metal mesh at the base of a flat bottomed bottle.Then place the fixed larvae on the mesh, followed by placing another mesh. Dehydrate the samples in an increasing ethanol concentration for 20 minutes each at four degrees Celsius. Then rinse the samples twice with propylene oxide at room temperature for five minutes each.Next, place the samples in a propylene oxide and epoxy resin solution, followed by pure epoxy resin for two hours at room temperature. For embedding, cut a polyethylene film into a big square and a small, hollow rectangular support. Glue the small square with the big square.Add sufficient epoxy resin at the center of the big square. Then turn on the stereoscope, and place the samples in the pre-positioned epoxy resin with the muscle side facing upwards. Add several drops of epoxy resin onto the samples, and cover them with polyethylene film.Place the samples for polymerization at increasing temperatures for 24 hours each. After polymerization, using a sharp blade, remove the excess parts of the larval body while retaining a trapezoid, including the A2 and A3 segment. Remove the trapezoid from the remaining larval body wall, and attach it to the resin-filled capsule with AB glue.Under a light microscope, confirm the position of the sixth and seventh muscles of the A2 and A3 segments, maintaining the tangent plane parallel to the sixth muscle. Then cut away one-fifth of the sample along the two sides of the A3 segment. Using a microtome, prepare an ultra thin section of the sample starting from the sixth muscle.After sectioning, attach as many slices as possible to each copper mesh. Use a transmission electron microscope to observe the samples. Among Lowicryl K4M resin and epoxy resin, Lowicryl K4M resin provided sharper and more easily observable results of the Drosophila body muscles.Confocal imaging demonstrated enhanced visualization of the neuromuscular junction between the sixth and seventh muscles. Transmission electron microscopy revealed bead-like neuromuscular junctions, offering improved synaptic structure information. The samples remained flat throughout the dehydration process due to the restriction of the metal meshes.Besides, the samples were polymerized between the plastic products, preventing the samples from folding. In the future, the TEM sample preparation method can be applied to brain and ventral nerve cord neuropil, thereby improving the potential of ultrastructural research.

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