We thank Gary Laevsky for helpful discussions on microscopy. This work was funded by NIH grants R01GM061107 and R01GM067758 to E.R.G.

Note: The procedure for muscle removal (Figure 1) is a modification of previously described methods for preparing larval fillets. The steps that precede and follow muscle removal are outlined briefly and the reader is referred to previous work 10, 11 for more detailed descriptions.
1. Dissect Larva in Cold Saline
<ol>
	<li>Prepare a working dilution of cold HL3.1 saline15 or cold Ca2+-free HL3.1 saline11 (Table 1). Place the larva in a silicone elastomer dish with just enough cold saline to cover the bottom of the dish. 
	NOTE: See Discussion regarding the choice of saline.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1x HL3.1 saline (pH 7.2)</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; 5 mM HEPES</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; 70 mM NaCl</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; 5 mM KCl</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; 1.5 mM CaCl2&#160;&#160; (omit for Ca2+-free saline)</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; 4 mM MgCl2</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; 10 mM NaHCO3</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; 5 mM trehalose</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; 115 mM sucrose</td>
		</tr>
		<tr>
			<td></td>
		</tr>
		<tr>
			<td>Filter sterilize and store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Note: Composition mimics insect hemolymph</td>
		</tr>
	</tbody>
</table>
Table 1. Composition of Cold Saline.
<ol start="2">
	<li>Position the larva with the ventral side up. The ventral surface of the larva can be identified by the abdominal dentical belts and the dorsal side by the primary larval tracheal tubes running from anterior to posterior. Stretch the larva in the anterior-posterior direction and pin the head and tail to a silicone elastomer dissecting dish using insect pins. Use fine dissecting scissors to cut along the ventral midline, beginning at one end and progressing toward the other. 
	NOTE: This orientation preserves the commonly studied dorsal cluster of da neurons.</li>
	<li>After the larva is cut open, pin the four corners to the dissecting dish as though opening a book. Use forceps to grab and remove internal organs including the CNS, gut, and trachea. Adjust insect pins so that the fillet is taut but not maximally stretched. 
	NOTE: Muscles are anchored to the body wall at segmental boundaries. Although muscles cover most of the body wall, they are absent in a narrow region near the dorsal midline.</li>
</ol>
2. Muscle Removal
<ol>
	<li>Locate the dorsal midline of the larva, where muscle tissue is absent. Position a single forceps prong such that it can be inserted underneath the muscle tissue in the flattest possible orientation.</li>
	<li>Starting at the dorsal midline, near the anterior boundary of the segment, carefully slide the forceps prong between the muscle and epidermis, taking care to minimize contact between the forceps and epidermis.</li>
	<li>Pull the forceps upwards to break the attachment of the muscle to the body wall at one anchor point. Repeat this process for the remaining hemi-segment(s) of interest. 
	NOTE: This protocol optimizes preservation of the posterior of each da neuron dendrite field. To preserve the anterior part of the dendrite field, it is best to insert the forceps prong at the posterior end of each segment.</li>
	<li>Re-adjust insect pins such that the larval fillet is maximally stretched in all directions.</li>
	<li>Fix the fillet while it is still pinned to the dissecting dish using cold, freshly prepared 4% formaldehyde in PBS for 25 min.</li>
	<li>Rinse 5 times in PBS.</li>
	<li>Use forceps to carefully pull away muscle tissue from the remaining anchor points, taking care to minimize contact with the epidermis.</li>
	<li>Unpin and remove the fillet from the dissecting dish to a 1.5 ml microcentrifuge tube. Perform all subsequent washing, blocking, and immunofluorescence staining steps, as previously described11.</li>
</ol>
3. Mount the Larval Fillet
<ol>
	<li>First remove the head and tail using fine dissecting scissors in order to make the sample as flat as possible. Mount the fillet on a coverslip in antifade mountant with the inner surface of the fillet facing the coverslip.</li>
	<li>Place a microscope slide on the coverslip and press gently to disperse the mounting medium. Flip the microscope slide over and seal the coverslip on the slide using nail polish. Slides can be stored at -20 &#176;C for at least one month.</li>
</ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+establish+dendrite-substrate+relationships+that+promote+dendritic+self-avoidance+and+patterning+in+Drosophila+sensory+neurons.">Integrins establish dendrite-substrate relationships that promote dendritic self-avoidance and patterning in Drosophila sensory neurons.</a> Neuron. 73 (1), 79-91 (2012).</li><li>Han, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+regulate+repulsion-mediated+dendritic+patterning+of+Drosophila+sensory+neurons+by+restricting+dendrites+in+a+2D+space.">Integrins regulate repulsion-mediated dendritic patterning of Drosophila sensory neurons by restricting dendrites in a 2D space.</a> Neuron. 73 (1), 64-78 (2012).</li><li>Parrish, J. Z., Xu, P., Kim, C. C., Jan, L. Y., Jan, Y. 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(57), e3111 (2011).</li><li>Gustafsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surpassing+the+lateral+resolution+limit+by+a+factor+of+two+using+structured+illumination+microscopy.">Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy.</a> J Microscopy. 198 (2), 82-87 (2000).</li><li>Ashdown, G. W., Cope, A., Wiseman, P. W., Owen, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+flow+quantified+beyond+the+diffraction+limit+by+spatiotemporal+image+correlation+of+structured+illumination+microscopy+data.">Molecular flow quantified beyond the diffraction limit by spatiotemporal image correlation of structured illumination microscopy data.</a> Biophys J. 107 (9), L21-L23 (2014).</li><li>Zhang, W., Yan, Z., Jan, L. Y., Jan, Y. 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The authors have nothing to disclose.

Here a protocol is described for manual removal of muscle tissue from Drosophila larval fillets. This protocol modifies previously described larval dissection techniques10,11. After the larva is dissected in a silicone elastomer dish, the dorsal midline is located. A single forceps prong, in its flattest possible orientation, is carefully inserted between the muscle tissue and the epidermis, near the dorsal midline. The forceps are gently pulled upwards to separate muscle tissue from one anchor point in each larval segment of interest. The larval fillet is then fixed in cold formaldehyde after which forceps are used again to pull away muscle from the remaining anchor points.
While it is tempting to perform the entirety of the muscle removal procedure post-fixation, we find that stretched muscle, once fixed, remains flattened and closely apposed to the epidermis even after detachment from an anchor point, rendering it difficult to manipulate without damaging the underlying tissue. Additionally, in our experience, fixed muscle tissue is brittle and tears easily, preventing its complete removal from the body wall. In contrast, when muscle is detached from one anchor point prior to fixation, it will contract towards the remaining anchor point during fixation, leaving tissue stubs that can be more easily grabbed by forceps and pulled from the larval body wall.
In order to best preserve samples, considerable care must be taken to minimize contact between the forceps and the epidermis during the muscle removal procedure. As a result, our protocol may add several minutes to previously described dissection techniques10,11. To avoid tissue degradation, the number of larvae that are dissected prior to fixation should be limited such that elapsed time is no more than 25 minutes. Additionally, we recommend testing multiple pairs of forceps &#8212; forceps of the same model may vary considerably in shape and sharpness&#160;&#8212; and adjusting the extent to which fillets are stretched prior to muscle removal in order to improve tissue preservation and dissection speed.
Lastly, we recommend experimenting with the use of Ca2+-free HL3.1 saline versus standard HL3.1 saline during larval dissection. In the absence of calcium, larval muscle contractions are greatly reduced. Thus, larval fillets may be easier to manipulate when dissected in Ca2+-free HL3.1 saline. However, we find that muscle contraction creates space between the muscle and epidermis and this can facilitate the insertion of forceps between these tissues while minimizing risk of damage to the epidermis. As a result, standard HL3.1 may be preferable for preserving the integrity of the epidermis and da neurons. We obtain good results with either saline and the choice is left to the user.
While our protocol increases the duration and complexity of previously described dissection techniques10,11, it offers several improvements that are useful for imaging larval sensory neurons and epidermal cells. First, it permits imaging of neuronally and epidermally expressed proteins that are otherwise obscured by muscle tissue. Second, it improves the fluorescence signal to noise ratio. Lastly, it enables use of super-resolution microscopy for superior discrimination of protein spatial relationships in da neurons and the epidermis. The improved imaging quality that we observe following muscle removal is likely the result of reduced photon absorption and scattering as well as reduced distance between the sample and the coverslip. Although not demonstrated here, muscle removal likely also improves antibody access to the epidermis and da neurons, improving the quality of immunofluorescence. Greater antibody accessibility may be particularly useful for immunofluorescence protocols performed in the absence of a tissue permeabilizing agent4.
The improvements gained by use of this protocol are likely to benefit studies of the neuronal and epidermal factors that regulate dendrite morphogenesis in sensory neurons. Furthermore, muscle removal has previously facilitated electrophysiological study of sensory neurons in the larval body wall and a modified version of this protocol may be useful for future studies that employ neurophysiological manipulation of larval sensory neurons. Lastly, a modified version of this protocol may be useful towards other applications such as the isolation of single da neurons for gene expression profiling.

Drosophila larval dendritic arborization (da) neurons provide a valuable model for studying neuronal development due to their amenability to genetic manipulation and the ease with which they can be imaged. These sensory neurons have been instrumental in the identification of numerous pathways that control dendrite morphogenesis1-3.
Four classes of da neurons (class I - IV) innervate the larval epidermis. These neurons lie between the basement membrane and the epidermis, with their dendrites forming largely two-dimensional arrays4,5. Of the four classes, class IV da neurons have the most highly branched arbors and, like sensory neurons of other animals, the elaboration of these arbors requires intrinsic factors as well as cues from neighboring tissues, particularly the epidermis, for their development6-9.
Studies to determine how such neuronal and extra-neuronal factors control dendrite morphogenesis benefit from the ability to detect protein expression in situ by immunofluorescence. The outer cuticle of the larva is impenetrable to antibodies, but this impediment is easily overcome by the preparation of larval fillets through well-established dissection methods10,11. However, the body wall muscle tissue that lies just interior to the basement membrane presents several challenges towards visualization of da neurons and epidermal cells. First, the muscle tissue, which lines most of the body wall, greatly obscures fluorescent signals emanating from neuronal or epidermal tissue. This substantially reduces the signal to noise ratio in the sample. Second, many relevant proteins may be expressed in the muscle tissue as well as in the neurons or epidermis. This muscle-derived fluorescence signal is likely to further obscure detection of a fluorescence signal from the neuron or epidermis. Finally, advances in microscopy technologies now permit imaging of samples at sub-diffraction resolution and may be especially helpful in discerning the localization of proteins that are expressed in neurons and surrounding epidermal cells12,13. However, imaging via super-resolution microscopy benefits from a strong signal to noise ratio and close proximity of the sample to the coverslip. In addition to reducing the signal to noise ratio, the larval body wall muscle distances da neurons from the coverslip, thereby limiting the improved image resolution that can be achieved with super-resolution microscopy methods. Besides challenges for immunofluorescence analysis, muscle tissue presents a barrier to electrophysiological recording from sensory neurons in the larval body wall. Its removal therefore benefits neurophysiological manipulation of sensory neurons14.
Here a method for manual removal of Drosophila larval muscle tissue is described. We demonstrate that our protocol permits immunofluorescence imaging of proteins that are otherwise obscured by muscle tissue, improves the signal to noise ratio for visualization of class IV da neurons, and enables the use of super-resolution microscopy to better discriminate spatial relationships of proteins and cellular structures in da neurons and the epidermis.

<table><tbody><tr><td>Dumont #5 tweezers</td><td>Electron Microscopy Sciences</td><td>72701-D</td><td></td></tr><tr><td>Micro Scissors, 8 cm, straight, 5 mm blades, 0.1 mm tips</td><td>World Precision Instruments</td><td>14003</td><td></td></tr><tr><td>Sylgard 184 silicone elastomer kit</td><td>Dow Corning</td><td>3097358-1004</td><td>for dissecting plates</td></tr><tr><td>Austerlitz insect pins, 0.1 mm</td><td>Fine Science Tools</td><td>26002-10</td><td></td></tr><tr><td>Fostec 8375 light source</td><td>Artisan Technology Group</td><td>62792-4</td><td></td></tr><tr><td>Zeiss Stemi 2000</td><td>Carl Zeiss Microscopy</td><td></td><td></td></tr><tr><td>Vectashield antifade mounting medium</td><td>Vector Laboratories</td><td>H-1000</td><td>for confocal microscopy</td></tr><tr><td>Prolong Diamond antifade mountant</td><td>Life Technologies</td><td>P36970</td><td>for structured illumination microscopy</td></tr><tr><td>Micro cover glass, 22 x 22 mm, No. 1.5</td><td>VWR</td><td>48366-227</td><td></td></tr><tr><td>Superfrost Plus microscope slides, 25 x 75 x 1.0 mm</td><td>Fisherbrand</td><td>12-550-15</td><td></td></tr><tr><td>Mouse anti-Coracle antibody</td><td>Developmental Studies Hybridoma Bank</td><td>C615.16</td><td>supernatant, dilute 1:50</td></tr><tr><td>Mouse anti-Discs large antibody</td><td>Developmental Studies Hybridoma Bank</td><td>4F3</td><td>supernatant, dilute 1:50</td></tr><tr><td>Rabbit anti-dsRed antibody</td><td>Clontech</td><td>632496</td><td>dilute 1:1,000</td></tr><tr><td>Goat anti-rabbit antibody, Alexa Fluor 568 conjugated</td><td>ThermoFisher Scientific</td><td>A-11011</td><td>dilute 1:1,000</td></tr><tr><td>Goat anti-mouse antibody, Alexa Fluor 488 conjugated</td><td>ThermoFisher Scientific</td><td>A-11001</td><td>dilute 1:500</td></tr></tbody></table>

removal of drosophila muscle tissue from larval fillets for immunofluorescence analysis of sensory neurons and epidermal cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

We demonstrate the utility of the muscle removal procedure for improving signal to noise ratio in immunofluorescence experiments to co-visualize the septate junction proteins Coracle (Cora) and Discs-large (Dlg) together with class IV da neurons labeled with the membrane marker CD4-tdTomato.
Cora has been previously used to identify tracts where da neuron dendrites are enclosed by epidermal cells and is one of many identified epidermal factors that have been studied in association with the morphogenesis and function of da neuron dendrites4,6-9,16. Immunostaining with anti-DsRed to detect the neurons (red) and anti-Cora antibody (green) was performed on larval fillets with muscle removed (muscle-off) and with muscle intact (muscle-on). For each condition, the posterior dendrite field of the class IV da neuron was imaged using a laser scanning confocal microscope. Images were obtained using identical imaging parameters for both conditions. We additionally imaged muscle-on fillets using increased laser power to compensate for interference from the muscle tissue.
In muscle-off samples, Cora could be clearly detected at epidermal cell boundaries, in intermittent tracts along class IV da neuron dendrites, and outlining the class IV da neuron soma (Figure 2C). In contrast, its localization to these domains was largely obscured in muscle-on samples, even when laser power was increased (Figure 2A-2B). In muscle-on samples, Cora was primarily visualized in muscle, trachea, and at the neuromuscular junction (NMJ), which become separated from the body wall as a result of the muscle removal procedure. The fluorescence signal emanating from these tissues obscured most of the Cora that localizes to epidermal cell boundaries and to dendrite tracts, except for in the narrow strip of the body wall near the dorsal midline, where muscle is absent. Cora that outlines the class IV da neuron soma was not visualized in muscle-on samples (Figure 2A-2B). Visualization of epidermally expressed proteins that also have high muscle expression, such as Dlg, is particularly problematic. In muscle-on samples, the distribution of Dlg in the epidermis was almost completely obscured by fluorescence from the muscle and NMJ (Figure 2D). After muscle removal, Dlg is readily detected at epidermal cell boundaries, in intermittent tracts along da neuron dendrites, and outlining the class IV da neuron soma (Figure 2E). Thus, muscle removal enables visualization of epidermally and neuronally expressed proteins otherwise obscured by muscle and associated tissues.
In addition, it was observed that the fluorescence intensity of the class IV da neuron marker appeared reduced in muscle-on fillets as compared to muscle-off fillets when imaging parameters were held constant between the two sample preparations (Figure 2A, 2C). With increased laser power, the apparent fluorescence intensity of the class IV da neuron marker could be matched to muscle-off samples but background noise was increased (Figure 2B, 2C). Therefore, muscle removal improves the signal to noise ratio in our samples.
Lastly, we tested whether the improvements gained by removing muscle tissue could be applied to imaging of class IV da neurons at sub-diffraction resolution. To do this, 3D structured illumination microscopy (SIM) imaging was performed, which achieves approximately 120 nm lateral resolution13. As with confocal imaging, we compared muscle-on and muscle-off fillets immunostained for Cora and class IV da neurons, first under identical imaging conditions and then after optimizing laser power, exposure time, and image processing for muscle-on samples. Similarly to confocal microscopy, a substantially reduced signal to noise ratio in muscle-on was observed as compared to muscle-off fillets (Figure 3A-3C). Additionally, image reconstruction appeared sharper in muscle-off samples. Compared to muscle-on samples, fewer apparent artifacts were observed and Cora dendrite tracts were more readily resolved (Figure 3B, 3C). Dendrite membranes could be measured at approximately 114 nm in width in muscle-off fillets but appeared to be 272 nm wide in muscle-on fillets (Figure 3D, 3E). Thus, our protocol enables the application of super-resolution microscopy to visualization of class IV da neurons.
<img alt="Figure 1" src="/files/ftp_upload/54670/54670fig1.jpg" /> 
Figure 1. Overview of Muscle Removal Procedure.&#160;A larva is dissected and pinned to a silicone elastomer dissecting dish. To remove muscle tissue, one forceps prong is inserted between the muscle and epidermal cell layer, starting from the dorsal midline near a segment boundary (2.2). The forceps is pulled upwards to break the attachment between the muscle and body wall (2.3). This is repeated for segments of interest and then the larva is fixed in formaldehyde (2.5). After fixation, forceps are used to separate the remaining muscle tissue from the body wall (2.7 - 2.8) The larval cuticle and epidermis are depicted in orange, class IV da neurons in green, and muscles in red. Insets in (2.2) and (2.7) represent cross-sectional views of a single larval hemi-segment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54670/54670fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54670/54670fig2.jpg" /> 
Figure 2. Muscle Removal Improves Confocal Imaging of da Neurons and the Epidermis.&#160;Immunofluorescence of Cora (green; A-C) or Dlg (green; D-E) was detected with muscle tissue (A, B, D) and without muscle tissue (C, E). Class IV da neurons were labeled using the GAL4&#39;477 driver to express UAS-CD4:tdTomato and detected with an anti-dsRed antibody (red). Muscle-on and muscle-off samples labeled for Cora were first imaged with a confocal microscope under identical conditions (A, C) and then with increased laser power to compensate for muscle interference (B). Muscle-on and muscle-off samples labeled for Dlg were imaged under individually optimized conditions. All images are confocal Z-projections. The middle (green) and bottom (red) panels show individual channels; the top panels are the merged channels. The boundary of the muscle tissue (dashed lines), the class IV da neuron soma (cyan arrows), Cora and Dlg dendrite tracts (white arrows), epidermal cell boundaries (magenta arrows), NMJ (yellow arrows), and trachea (orange arrows) are indicated. Scale bar = 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54670/54670fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54670/54670fig3.jpg" /> 
Figure 3. Muscle Removal Enables Super-resolution of Imaging of da Neurons and Epidermal Cells.&#160;Immunofluorescence detection of Cora (green) in class IV da neurons with muscle intact (A, B) and after muscle removal (C). Class IV da neurons were labeled as in Figure 2. Muscle-on and muscle-off samples were imaged with a structured illumination microscope first under identical conditions (A, C); the muscle-on sample in (A) was then re-imaged with increased laser power and exposure times to compensate for muscle interference (B). All images are Z-projections. The middle (green) and bottom (red) panels of A-C show individual channels. The top panel of A-C is a merge. Epidermal cell boundaries (magenta arrows), Cora dendrite tracts (white arrows), and an image reconstruction artifact (yellow arrowhead) are indicated. Insets in the bottom panels of B and C correspond to D and E, respectively. The apparent width of the dendrite membrane is shown in muscle-on (D) and muscle-off (E) samples. Scale bars = 3 &#181;m (A-C) and 0.5 &#181;m (D, E). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54670/54670fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells at JoVE.com

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

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JoVE Journal

Neuroscience

8.9K Views.  Princeton University. Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

Video: Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and mechanobiology, which all influence muscle development and myofibrillogenesis. Omics data, such as those generated by mass spectrometry or deep sequencing, can provide important mechanistic insights into these biological processes. For such approaches, it is beneficial to analyze tissue-specific samples to increase both selectivity and specificity of the omics fingerprints. Here we present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched muscle samples for omics applications. We first describe how to dissect flight muscles at early pupal stages (&#60;48 h after puparium formation [APF]), when the muscles are discernable by green fluorescent protein (GFP) labeling. We then describe how to dissect muscles from late pupae (&#62;48 h APF) or adults, when muscles are distinguishable under a dissecting microscope. The accompanying video protocol will make these technically demanding dissections more widely accessible to the muscle and Drosophila research communities. For RNA applications, we assay the quantity and quality of RNA that can be isolated at different time points and with different approaches. We further show that Bruno1 (Bru1) is necessary for a temporal shift in myosin heavy chain (Mhc) splicing, demonstrating that dissected muscles can be used for mRNA-Seq, mass spectrometry, and reverse transcription polymerase chain reaction (RT-PCR) applications. This dissection protocol will help promote tissue-specific omics analyses and can be generally applied to study multiple biological aspects of myogenesis.

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study transcriptional regulation, alternative splicing, metabolism, and mechanobiology. We present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched samples ideal for proteomics and deep-sequencing. These samples can offer important mechanistic insights into diverse aspects of muscle development.

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and ...

Genetics

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Biology

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

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

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The well characterized larval neuromuscular junction is often used for such studies. However, rapid larval development followed by muscle histolysis and nervous system remodeling during metamorphosis makes this model problematic for the study of slow age-dependent degenerative changes like those occurring in amyotrophic lateral sclerosis. Alternatively, adult flies live for 90 days and the adult leg can be used to study motor neuron changes over the course of adult lifespan using in vivo fluorescent imaging through the cuticle. Here, we describe a leg dissection technique coupled with immunocytochemistry, which allows for the study of molecular changes at the neuromuscular junction of identified adult leg motor neurons. These techniques can be coupled with a myriad of antibodies labeling both pre- and post-synaptic structures. Together these procedures allow a more complete characterization of slow age-dependent changes in adult flies and can be applied across multiple motor neuron disease models.

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

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.

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The ...

Viewing Microtubule Networks in Drosophila Neuromuscular Junctions and Muscle Cells

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

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

Author Spotlight: Understanding Microtubule Network in Drosophila Neuromuscular Junctions 

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.

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with ...

Single-Neuron Ablation in Drosophila for Studying Nervous System Adaptation

Laser Cell Ablation in Intact Drosophila Larvae Reveals Synaptic Competition

The protocol describes single-neuron ablation with a 2-photon laser system in the central nervous system (CNS) of intact Drosophila melanogaster larvae. Using this non-invasive method, the developing nervous system can be manipulated in a cell-specific manner. Disrupting the development of individual neurons in a network can be used to study how the nervous system can compensate for the loss of synaptic input. Individual neurons were specifically ablated in the giant fiber system of Drosophila, with a focus on two neurons: the presynaptic giant fiber (GF) and the postsynaptic tergotrochanteral motor neuron (TTMn). The GF synapses with the ipsilateral TTMn, which is crucial to the escape response. Ablating one of the GFs in the 3rd instar brain, just after the GF starts axonal growth, permanently removes the cell during the development of the CNS. The remaining GF reacts to the absent neighbor and forms an ectopic synaptic terminal to the contralateral TTMn. This atypical, bilaterally symmetric terminal innervates both TTMns, as demonstrated by dye coupling, and drives both motor neurons, as demonstrated by electrophysiological assays. In summary, the ablation of a single interneuron demonstrates synaptic competition between a bilateral pair of neurons that can compensate for the loss of one neuron and restore normal responses to the escape circuit.

Author Spotlight: Investigating the Mechanisms of Neural Circuit Assembly and Synapse Formation in Drosophila

This protocol demonstrates the laser cell ablation of individual neurons in intact Drosophila larvae. The method enables the study of the effect of reducing competition between neurons in the developing nervous system.

The protocol describes single-neuron ablation with a 2-photon laser system in the central nervous system (CNS) of intact Drosophila melanogaster larvae. ...

Drosophila Late Pupa Indirect Flight Muscle (IFM) Dissection: A Method for High-Throughput Tissue Collection

Source: Kao, S. Y., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/60309/" target="_blank">Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches</a>. J. Vis. Exp. (2019).
The Drosophila flight muscles are used as a model system for muscle development and physiology. This video describes the flight musculature of adults and highlights a protocol to dissect developing indirect flight muscles that are suitable for RNA sequencing from pupae.

Drosophila Rozwarstwienie mięśni lotu pośredniego późnej poczwarki (IFM): metoda wysokoprzepustowego pobierania tkanek

Source: Kao, S. Y., et al. Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches. J. Vis. Exp. (2019).
The Drosophila flight muscles ...

JoVE Encyclopedia of Experiments

Drosophila melanogaster (fruit fly)

Pupa

Larval Fillet Preparation: A Method to Visualize Intact Sensory Neurons and Associated Epidermal Cells

Source: Tenenbaum, C. M., Gavis, E. R. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/54670/" target="_blank">Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells</a>. J. Vis. Exp. (2016).
This video describes how to prepare larval fillets used to visualize sensory dendritic arborization (da) neurons and their associated epidermal cells. The featured protocol shows in detail how to remove muscle tissue from the body wall while preserving the cells&#39; morphology, which improves immunostaining and visualization of the otherwise obscured da neurons and epidermal cells.

Przygotowanie filetów larwalnych: metoda wizualizacji nienaruszonych neuronów czuciowych i związanych z nimi komórek naskórka

Source: Tenenbaum, C. M., Gavis, E. R. Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and ...

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

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Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila Larval NMJ Dissection

Drosophila Larval NMJ Immunohistochemistry

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

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

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

Laser Cell Ablation in Intact Drosophila Larvae Reveals Synaptic Competition

Drosophila Rozwarstwienie mięśni lotu pośredniego późnej poczwarki (IFM): metoda wysokoprzepustowego pobierania tkanek

Przygotowanie filetów larwalnych: metoda wizualizacji nienaruszonych neuronów czuciowych i związanych z nimi komórek naskórka