Name: Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae (Video) | JoVE Journal
Uploaded: 2023-09-15T14:00:00.000+00:00
Duration: 5 min 54 s
Description: Learn an optimized TEM sample preparation method for visualizing neuromuscila junctions in Drosophila larvae. This protocol prevents sample curling using metal meshes, enabling improved ultrastructural analysis of synaptic structures.

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5 minutes

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.
1. Dissection, fixation and placement procedure
<ol>
	<li>Dissection
	<ol>
		<li>Dissect the wandering late-3rd-instar larvae in Jan solution and obtain the entire body wall based on the standard procedures17. Prepare Jan&#39;s solution as following: 128 mM NaCl, 2 mM KCl, 4 mM MgCl2, 35mM sucrose, 5mM HEPES, pH 7.4 
		NOTE: The body wall of Drosophila larvae refers to the outer covering or integument of the larval stage of the fruit fly Drosophila. This includes the cuticle, epidermal cells, and underlying tissues that form the larval exoskeleton. The body wall provides structural support and protection and serves as a barrier between the larval body and the external environment17.</li>
	</ol>
	</li>
	<li>Fixation
	<ol>
		<li>Fix the larval body wall in the dissection chamber at 4 &#176;C overnight with the fixative. To prepare the fixative, mix 2% glutaraldehyde and 2% formaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4)</li>
		<li>Rinse the samples several times with sodium cacodylate buffer.</li>
		<li>Prepare 1% OsO4 in cacodylate buffer. Fix the samples in 1% OsO4 for 2 h, followed by several rinses in distilled water. 
		NOTE: Dual fixation with aldehydes and OsO4 provides better protection of cellular ultrastructure. Besides, OsO4 can stain the sample tissue, allowing for the contrast between the light and the sample to be more apparent18.</li>
		<li>Stain the Drosophila neuromuscular junction samples with 2% saturated uranyl acetate for 2 h, followed by several rinses with deionized water (Figure 1A).</li>
	</ol>
	</li>
	<li>Sample placement
	<ol>
		<li>Cut two round pieces of metal mesh of similar size (pore size 270 &#181;m) with scissors.</li>
		<li>Place a metal mesh (270 &#181;m) at the base of a flat-bottomed bottle in advance.</li>
		<li>Place the fixed larva samples on the mesh and then place another mesh on them so that the samples are placed between the two meshes.</li>
	</ol>
	</li>
</ol>
2. Dehydration
NOTE In this paper, the ethanol gradient dehydration method was used.
<ol>
	<li>Dehydrate the samples in an increasing concentration gradient of ethanol sequentially (50%, 70%, 85%, 95% once each, 100% twice), each for 20 min at 4 &#176;C.</li>
	<li>Rinse the samples twice with propylene oxide at room temperature for 5 min each time (Figure 1C).</li>
</ol>
3. Infiltration
NOTE: Infiltration is one of the necessary steps for the TEM sample preparation. Infiltration allows for the gradual replacement of dehydrating medium with the embedding medium in the sample to allow filling of the cell interstice with the embedding medium19.
<ol>
	<li>Place the samples in the mixed solution of propylene oxide and epoxy resin at a ratio of 1:1 and 1:2 for 1 h each, followed by pure epoxy resin for 2 h at room temperature.</li>
</ol>
4. Embedding
<ol>
	<li>Prepare a polyethylene film and cut it into a big square (approximately 5 &#215; 5 cm) and a small hollow rectangular support (approximately 2 &#215; 2 cm). Glue the small square with the big square.</li>
	<li>Pre-position sufficient epoxy resin at the center of the big square.</li>
	<li>Place the samples in the pre-positioned epoxy resin with the muscle side facing upwards. Use a stereoscope.</li>
	<li>Add several drops of resin to the samples and then cover them with PE film (a little larger than the spacer) (Figure 1D-E).</li>
	<li>Polymerize the samples at 37 &#176;C, 42 &#176;C and 60 &#176;C for 24 h respectively (Figure 1F).</li>
</ol>
5. Sectioning procedure
<ol>
	<li>Remove the excess parts of the larval body wall with a sharp blade and retain a trapezoid, including the A2/A3 segment. 
	NOTE: The top line should be near the 13th muscle, and the baseline should be away from the sample to the epoxy resin. The height between the topline and baseline is approximately 5-8 mm.</li>
	<li>Remove the trapezoid from the remainder of the sample and adhere it to the resin-filled capsule with the A/B glue.</li>
	<li>Confirm the position of the 6th and 7th muscles of A2 and A3 segments under the light microscopy (Figure 1G) and maintain the tangent plane parallel to the 6th muscle.</li>
	<li>Cut away one-fifth of the sample along the two sides of the A3 segment. 
	NOTE: Remove most of the 6th muscle, and retain 1/3 of the width of the 7th muscle.</li>
	<li>Using an ultrathin microtome, prepare an ultrathin section of the sample starting with the 6th muscle. 
	NOTE: Each slice is approximately 90 nm thick.</li>
	<li>Attached as many slices as possible to each copper mesh.</li>
	<li>Perform transmission electron microscopy as described previously16.</li>
</ol>

<ol><li>Acevedo, N. C., Marangoni, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanostructured+fat+crystal+systems%5BTitle%5D)+AND+%22Annual+Review+of+Food+Science+and+Technology%22%5BJournal%5D)">Nanostructured fat crystal systems.</a> Annual Review of Food Science and Technology. 6, 71-96 (2015).</li>
<li>Schorb, M., Haberbosch, I., Hagen, W. J. H., Schwab, Y., Mastronarde, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Software+tools+for+automated+transmission+electron+microscopy%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Software tools for automated transmission electron microscopy.</a> Nature Methods. 16 (6), 471-477 (2019).</li>
<li>Winans, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+genomic+investigation+of+ecological+differentiation+between+free-living+and+Drosophila-associated+bacteria%5BTitle%5D)+AND+%22Molecular+Ecology%22%5BJournal%5D)">A genomic investigation of ecological differentiation between free-living and Drosophila-associated bacteria.</a> Molecular Ecology. 26 (17), 4536-4550 (2017).</li>
<li>Phelps, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reconstruction+of+motor+control+circuits+in+adult+Drosophila+using+automated+transmission+electron+microscopy%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Reconstruction of motor control circuits in adult Drosophila using automated transmission electron microscopy.</a> Cell. 184 (3), 759-774 (2021).</li>
<li>Cardona, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+integrated+micro-+and+macroarchitectural+analysis+of+the+Drosophila+brain+by+computer-assisted+serial+section+electron+microscopy%5BTitle%5D)+AND+%22PLoS+Biology%22%5BJournal%5D)">An integrated micro- and macroarchitectural analysis of the Drosophila brain by computer-assisted serial section electron microscopy.</a> PLoS Biology. 8 (10), (2010).</li>
<li>Belalcazar, H. M., Hendricks, E. L., Zamurrad, S., Liebl, F. L. W., Secombe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+histone+demethylase+KDM5+is+required+for+synaptic+structure+and+function+at+the+Drosophila+neuromuscular+junction%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">The histone demethylase KDM5 is required for synaptic structure and function at the Drosophila neuromuscular junction.</a> Cell Reports. 34 (7), 108753(2021).</li>
<li>Banerjee, S., Venkatesan, A., Bhat, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurexin%2C+neuroligin+and+wishful+thinking+coordinate+synaptic+cytoarchitecture+and+growth+at+neuromuscular+junctions%5BTitle%5D)+AND+%22Molecular+Cell+and+Neurosciences%22%5BJournal%5D)">Neurexin, neuroligin and wishful thinking coordinate synaptic cytoarchitecture and growth at neuromuscular junctions.</a> Molecular Cell and Neurosciences. 78, 9-24 (2017).</li>
<li>Guangming, G., Junhua, G., Chenchen, Z., Yang, M., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurexin+and+neuroligins+maintain+the+balance+of+ghost+and+satellite+boutons+at+the+Drosophila+neuromuscular+junction%5BTitle%5D)+AND+%22Frontiers+in+Neuroanatomy%22%5BJournal%5D)">Neurexin and neuroligins maintain the balance of ghost and satellite boutons at the Drosophila neuromuscular junction.</a> Frontiers in Neuroanatomy. 14, 19(2020).</li>
<li>Jia, X. X., Gorczyca, M., Budnik, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrastructure+of+neuromuscular+junctions+in+Drosophila%3A+comparison+of+wild+type+and+mutants+with+increased+excitability%5BTitle%5D)+AND+%22Journal+of+Neurobiology%22%5BJournal%5D)">Ultrastructure of neuromuscular junctions in Drosophila: comparison of wild type and mutants with increased excitability.</a> Journal of Neurobiology. 24 (8), 1025-1044 (1993).</li>
<li>Ashley, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retrovirus-like+gag+protein+Arc1+binds+RNA+and+traffics+across+synaptic+boutons%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Retrovirus-like gag protein Arc1 binds RNA and traffics across synaptic boutons.</a> Cell. 172 (1-2), 262-274 (2018).</li>
<li>Eaton, B. A., Fetter, R. D., Davis, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynactin+is+necessary+for+synapse+stabilization%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Dynactin is necessary for synapse stabilization.</a> Neuron. 34 (5), 729-741 (2002).</li>
<li>Featherstone, D. E., Rushton, E., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developmental+regulation+of+glutamate+receptor+field+size+by+nonvesicular+glutamate+release%5BTitle%5D)+AND+%22Nature+Neurosciences%22%5BJournal%5D)">Developmental regulation of glutamate receptor field size by nonvesicular glutamate release.</a> Nature Neurosciences. 5 (2), 141-146 (2002).</li>
<li>Wagner, N., Laugks, U., Heckmann, M., Asan, E., Neuser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aging+Drosophila+melanogaster+display+altered+pre-+and+postsynaptic+ultrastructure+at+adult+neuromuscular+junctions%5BTitle%5D)+AND+%22Journal+of+Comparative+Neurology%22%5BJournal%5D)">Aging Drosophila melanogaster display altered pre- and postsynaptic ultrastructure at adult neuromuscular junctions.</a> Journal of Comparative Neurology. 523 (16), 2457-2475 (2015).</li>
<li>Atwood, H. L., Govind, C. K., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+ultrastructure+of+synaptic+terminals+on+ventral+longitudinal+abdominal+muscles+in+Drosophila+larvae%5BTitle%5D)+AND+%22Journal+of+Neurobiology%22%5BJournal%5D)">Differential ultrastructure of synaptic terminals on ventral longitudinal abdominal muscles in Drosophila larvae.</a> Journal of Neurobiology. 24 (8), 1008-1024 (1993).</li>
<li>McCabe, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+BMP+homolog+Gbb+provides+a+retrograde+signal+that+regulates+synaptic+growth+at+the+Drosophila+neuromuscular+junction%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">The BMP homolog Gbb provides a retrograde signal that regulates synaptic growth at the Drosophila neuromuscular junction.</a> Neuron. 39 (2), 241-254 (2003).</li>
<li>Guangming, G., Mei, C., Chenchen, Z., Wei, X., Junhua, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+analysis+method+of+neuromuscular+junction+in+Drosophila.+larvae+by+transmission+electron+microscopy%5BTitle%5D)+AND+%22Anatomical+Science+International%22%5BJournal%5D)">Improved analysis method of neuromuscular junction in Drosophila. larvae by transmission electron microscopy.</a> Anatomical Science International. 97 (1), 147-154 (2022).</li>
<li>Brent, J. R., Werner, K. M., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drosophila+larval+NMJ+dissection%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Drosophila larval NMJ dissection.</a> Journal of Visualized Experiments. (24), e1107(2009).</li>
<li>Lovrić, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multimodal+imaging+of+chemically+fixed+cells+in+preparation+for+NanoSIMS%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">Multimodal imaging of chemically fixed cells in preparation for NanoSIMS.</a> Analytical Chemistry. 88 (17), 8841-8848 (2016).</li>
<li>Woods, P. S., Ledbetter, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+organelles+at+uncoated+cryofractured+surfaces+as+viewed+with+the+scanning+electron+microscope%5BTitle%5D)+AND+%22Journal+of+Cell+Sciences%22%5BJournal%5D)">Cell organelles at uncoated cryofractured surfaces as viewed with the scanning electron microscope.</a> Journal of Cell Sciences. 21 (1), 47-58 (1976).</li>
<li>Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Properties+of+the+larval+neuromuscular+junction+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22Journal+of+Physiology%22%5BJournal%5D)">Properties of the larval neuromuscular junction in Drosophila melanogaster.</a> Journal of Physiology. 262 (1), 189-214 (1976).</li>
<li>Prokop, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organization+of+the+efferent+system+and+structure+of+neuromuscular+junctions+in+Drosophila%5BTitle%5D)+AND+%22International+Review+in+Neurobiology%22%5BJournal%5D)">Organization of the efferent system and structure of neuromuscular junctions in Drosophila.</a> International Review in Neurobiology. 75, 71-90 (2006).</li>
<li>Lloyd, T. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+p150%28Glued%29+CAP-Gly+domain+regulates+initiation+of+retrograde+transport+at+synaptic+termini%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">The p150(Glued) CAP-Gly domain regulates initiation of retrograde transport at synaptic termini.</a> Neuron. 74 (2), 344-360 (2012).</li>
<li>Budnik, V., Zhong, Y., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphological+plasticity+of+motor+axons+in+Drosophila+mutants+with+altered+excitability%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Morphological plasticity of motor axons in Drosophila mutants with altered excitability.</a> Journal of Neuroscience. 10 (11), 3754-3768 (1990).</li>
<li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuroligin+4+regulates+synaptic+growth+via+the+bone+morphogenetic+protein+%28BMP%29+signaling+pathway+at+the+Drosophila+neuromuscular+junction%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Neuroligin 4 regulates synaptic growth via the bone morphogenetic protein (BMP) signaling pathway at the Drosophila neuromuscular junction.</a> Journal of Biological Chemistry. 292 (44), 17991-18005 (2017).</li>
<li>Wu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+pre-synaptic+function+of+shank+protein+in+Drosophila%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">A pre-synaptic function of shank protein in Drosophila.</a> Journal of Neuroscience. 37 (48), 11592-11604 (2017).</li>
<li>Gan, G., Lv, H., Xie, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphological+identification+and+development+of+neurite+in+Drosophila+ventral+nerve+cord+neuropil%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Morphological identification and development of neurite in Drosophila ventral nerve cord neuropil.</a> PLoS One. 9 (8), 105497(2014).</li>
</ol>

The authors have nothing to disclose.

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><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 W&lt;sup&gt;1118&lt;/sup&gt;, and were reared according to standard culture methods</td></tr><tr><td>Flat-bottomed glass test tubes</td><td>Haimen Chenxing Experimental equipment Company&amp;nbsp;</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>&amp;nbsp;Agar Scientific</td><td>Cat# 1924B</td><td>The embedding resins are based on a highly cross-linked acrylate and methacrylate formula&amp;nbsp;</td></tr><tr><td>K4M resin (monomer B)</td><td>&amp;nbsp;Agar Scientific</td><td>Lot# 631557</td><td>Resin Monomer</td></tr><tr><td>Polyvinyl film</td><td>Haimen Chenxing Experimental equipment Company&amp;nbsp;</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&amp;nbsp;</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

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-...

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.

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

optimizing-sample-preparation-process-for-transmission-electron

Research

JoVE Journal

Neuroscience

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The goal of this procedure is to dissect the dorsal longitudinal muscle (DLM) tissue to assess the structural integrity of DLM neuromuscular junctions (NMJs) in neurodegenerative disease models using Drosophila melanogaster.

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

We describe a dissection technique that preserves the architecture of the neuromuscular junction and enables a detailed immunocytochemical study of motor neurons in the adult Drosophila leg.

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

This protocol presents a practical method using Methanocaldococcus jannaschii (MjsHSP16.5) as an example to address uneven particle distribution through sample preparation optimization, providing a reference for researchers to efficiently elucidate macromolecular structures using cryogenic electron microscopy (cryo-EM).

Optimizing Sample Preparation for Cryogenic Electron Microscopy

Learn an optimized TEM sample preparation method for visualizing neuromuscila junctions in Drosophila larvae. This protocol prevents sample curling using metal meshes, enabling improved ultrastructural analysis of synaptic structures.

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

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