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)....

<ol>
	<li>Jan, Y. N., Jan, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Branching+out:+mechanisms+of+dendritic+arborization.">Branching out: mechanisms of dendritic arborization.</a> Nat Rev Neurosci. 11 (5), 316-328 (2010).</li><li>Corty, M. M., Matthews, B. J., Grueber, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecules+and+mechanisms+of+dendrite+development+in+Drosophila.">Molecules and mechanisms of dendrite development in Drosophila.</a> Development. 136 (7), 1049-1061 (2009).</li><li>Parrish, J. Z., Emoto, K., Kim, M. D., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+that+regulate+establishment,+maintenance,+and+remodeling+of+dendritic+fields.">Mechanisms that regulate establishment, maintenance, and remodeling of dendritic fields.</a> Annu Rev Neurosci. 30, 399-423 (2007).</li><li>Kim, M. E., Shrestha, B. R., Blazeski, R., Mason, C. A., Grueber, W. 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. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microRNA+bantam+functions+in+epithelial+cells+to+regulate+scaling+growth+of+dendrite+arbors+in+Drosophila+sensory+neurons.">The microRNA bantam functions in epithelial cells to regulate scaling growth of dendrite arbors in Drosophila sensory neurons.</a> Neuron. 63 (6), 788-802 (2009).</li><li>Jiang, N., Soba, P., Parker, E., Kim, C. C., Parrish, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microRNA+bantam+regulates+a+developmental+transition+in+epithelial+cells+that+restricts+sensory+dendrite+growth.">The microRNA bantam regulates a developmental transition in epithelial cells that restricts sensory dendrite growth.</a> Development. 141 (13), 2657-2668 (2014).</li><li>Meltzer, S., 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=Epidermis-derived+Semaphorin+promotes+dendrite+self-avoidance+by+regulating+dendrite-substrate+adhesion+in+Drosophila+sensory+neurons.">Epidermis-derived Semaphorin promotes dendrite self-avoidance by regulating dendrite-substrate adhesion in Drosophila sensory neurons.</a> Neuron. 89 (4), 741-755 (2016).</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=Epidermal+cells+are+the+primary+phagocytes+in+the+fragmentation+and+clearance+of+degenerating+dendrites+in+Drosophila.">Epidermal cells are the primary phagocytes in the fragmentation and clearance of degenerating dendrites in Drosophila.</a> Neuron. 81 (3), 544-560 (2014).</li><li>Brent, J. R., Werner, K. M., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+larval+NMJ+dissection.">Drosophila larval NMJ dissection.</a> J Vis Exp. (24), (2009).</li><li>Karim, M. R., Moore, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+analysis+of+Drosophila+larval+peripheral+sensory+neuron+dendrites+and+axons+using+genetic+mosaics.">Morphological analysis of Drosophila larval peripheral sensory neuron dendrites and axons using genetic mosaics.</a> J Vis Exp. (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. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sound+response+mediated+by+the+TRP+channels+NOMPC,+NANCHUNG,+and+INACTIVE+in+chordotonal+organs+of+Drosophila+larvae.">Sound response mediated by the TRP channels NOMPC, NANCHUNG, and INACTIVE in chordotonal organs of Drosophila larvae.</a> PNAS. 10 (33), 13612-13617 (2013).</li><li>Feng, Y., Ueda, A., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+minimal+hemolymph-like+solution,+HL3.1,+for+physiological+recordings+at+the+neuromuscular+junctions+of+normal+and+mutant+Drosophila+larvae.">A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae.</a> J Neurogenet. 18 (2), 377-402 (2004).</li><li>Babcock, D. T., Landry, C., Galko, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+signaling+mediates+UV-induced+nociceptive+sensitization+in+Drosophila+larvae.">Cytokine signaling mediates UV-induced nociceptive sensitization in Drosophila larvae.</a> Curr Biol. 19 (10), 799-806 (2009).</li></ol>

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 ...

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 border="0" cellpadding="0" cellspacing="0" width="1299">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Dumont #5 tweezers</td>
			<td style="width:304px;">Electron Microscopy Sciences</td>
			<td style="width:151px;">72701-D</td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Sylgard 184 silicone elastomer kit</td>
			<td>Dow Corning</td>
			<td>3097358-1004</td>
			<td>for dissecting plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Austerlitz insect pins, 0.1 mm</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fostec 8375 light source</td>
			<td>Artisan Technology Group</td>
			<td>62792-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeiss Stemi 2000</td>
			<td>Carl Zeiss Microscopy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield antifade mounting medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td>for confocal microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prolong Diamond antifade mountant</td>
			<td>Life Technologies</td>
			<td>P36970</td>
			<td>for structured illumination microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro cover glass, 22x22 mm, No. 1.5</td>
			<td>VWR</td>
			<td>48366-227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfrost Plus microscope slides, 25 x 75 x 1.0 mm</td>
			<td>Fisherbrand</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti-Coracle antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>C615.16</td>
			<td>supernatant, dilute 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti-Discs large antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>4F3</td>
			<td>supernatant, dilute 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit anti-dsRed antibody</td>
			<td>Clontech</td>
			<td>632496</td>
			<td>dilute 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-rabbit antibody, Alexa Fluor 568 conjugated</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11011</td>
			<td>dilute 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 st...

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 ...

The overall goal of this procedure is to remove muscle tissue from a drosophila larva in preparation for immunofluorescence analysis of larval dendritic arborization or da neurons and epidermal tissue. Today, we are going to demonstrate a method that improves visualization of neuronal and extra neuronal factors involved in dendrite morphogenesis. The main advantage of this technique is that it permits immunofluorescence imaging of proteins, that are otherwise obscured by muscle tissue, improve signal to noise ratio, and enables the use of super resolution microscopy.To begin, add enough cold saline to a silicone elastomer dish to cover the bottom surface. Add the subject larva to the dish and place it under the stereo microscope. Ensuring proper placement of light.Position the larva with the ventral side up, which can be identified by the abdominal denticle belts. Stretch the larva in the anterior posterior direction, and using insect pins, pin the head and tail to the bottom of the dish. With a pair of fine dissecting scissors, cut along the ventral midline of the larva from one end to the other.Spread the larva open and pin the cuticle tissue at the four corners so that it lies flat. Using forceps, remove the internal organs, including the CNS, gut, and trachea. Adjust the insect pins so that the filet is taught but not maximally stretched.Locate the dorsal midline of the larva, where muscle tissue is absent, and then position a forceps prong at the interior of this boundary. Slide the prong between the muscle and epidermis, taking care to minimize contact with the epidermis To minimize damage to the epidermis, dissect with the flattest possible prongs, and try dissecting in calcium containing saline. That could help create space between the muscle and the epidermis.Next, 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 muscle segments of interest. Readjust the insect pins to maximally stretch the larval fillets in all directions.To fix the filet, first remove the saline solution, and then add cold freshly prepared 4%formaldehyde in PBS, to cover the bottom of the dish. Incubate at room temperature for 25 minutes. Rinse the filet five times in PBS to remove excess formaldehyde.Next, use forceps to carefully pull away the muscle tissue from the remaining anchor points. Again, minimizing contact with the epidermis. Finally, unpin the filet and remove it from the dissecting dish.Place it into a clean 1.5 milliliter microcentrifuge tube. Further washing, blocking and immunostaining should be carried out in this tube. To mount the filet for imaging, remove the sample from the microcentrifuge tube and orient it inner surface down onto a cover slip in a drop of PBS.Cut off the head and tail. And then remove the PBS by absorbing it with a laboratory wipe. Add one drop of Antifade Mountant.Place a microscope slide onto the cover slip, and then press gently using a laboratory wipe to disperse the mounting medium. Flip the side over and apply clear nail polish at each edge to seal the cover slip. These confocal images show immunofluorescence experiments to visualize the septate junction protein, Coracle, or Cora, and da neurons.Here, images of muscle removed and muscle intact tissues were taken under the same laser power conditions. In muscle intact tissue, laser power was increased to compensate for muscle interference, and produce a comparable image. In images obtained from muscle removed fillets, cora could be clearly detected at epidermal cell boundaries in intermittent tracks along class four da neuron dendrites.And outlining the neuron, soma. In contrast, cora localization at these domains was barely detectable in muscle on samples. Here, cora was largely visualized in the muscle, trachea, and neuromuscular junctions, and fluorescence from these tissues obscured most epidermal cora.Samples were also imaged using 3D structured illumination microscopy to determine whether muscle removal improved visualization at higher resolution. Again, the muscle intact samples needed visualization with increased power and exposure time to compensate for muscle interference. Image resolution appeared sharper in muscle off samples, fewer artifacts were observed, and cora staining of dendrite tracks was more readily resolved.Dendrite membranes could also be measured at 114 nanometers in the muscle off condition. Whereas they appear to be 272 nanometers wide in muscle on filets. While performing this procedure, it's important to limit the number of larva dissected prior to fixation, so that the elapsed time is no more than 25 minutes.After watching this video, you should have a good understanding of how to remove muscle tissue from a drosophila larva. This protocol will assist with immunofluorescence analysis of larval sensory neurons and the epidermal cells they innovate. Thanks for watching and good luck with your experiments.

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8.8K Görüntüleme.  Princeton University. The overall goal of this procedure is to remove muscle tissue from a drosophila larva in preparation for immunofluorescence analysis of larval dendritic arborization or da neurons and epidermal tissue. Today, we are going to demonstrate a method that improves visualization of neuronal and extra neuronal factors involved in dendrite morphogenesis. The main advantage of this technique is that it permits immunofluorescence imaging of proteins, that are otherwise obscured by muscle tissue, improv...