This work was supported by grants from the National Natural Science Foundation of China (31671422, 31529004, and 31601112), the 111 Project (D18010), the Local Innovative and Research Teams Project of Guangdong Perl River Talents Program (2017BT01S155), and the China Postdoctoral Science Foundation (2018M640767).

1. Egg Laying
<ol>
	<li>Prepare the standard cornmeal food for egg laying. 
	NOTE: For the recipe and cooking procedure for standard cornmeal food used here, see the previously published details15.</li>
	<li>Prepare fresh yeast paste by adding 6 mL of distilled water to 4 g of active dried yeast in a 50 mL centrifugal tube. Use a spatula to mix and make a paste.</li>
	<li>Make egg-laying bottles by filling the cornmeal food into the bottles and spread approximately 1 g of yeast paste onto the surface of the cornmeal food with a spatula.</li>
	<li>Place the flies of desired genotypes in an egg-laying bottle and place it in an incubator with a constant temperature of 25 &#176;C and humidity of 60%. 
	NOTE: Ideal crosses usually consist of 150 virgin flies and at least 75 males. Keeping flies in dark by placing a lightproof box over the egg-laying bottles will increase the reproduction rate.</li>
	<li>Before egg collection, let flies lay eggs for 1 h to allow removal of all old eggs that remain stored in the female oviducts.</li>
	<li>Let flies lay eggs in a new egg-laying bottle for 1 h and remove the adults from the bottle. 
	NOTE: Control the egg-laying time to minimize the developmental range in order to obtain larvae within a precisely controlled developmental stage.</li>
	<li>Allow the eggs to develop for 84 h into the third instar larvae in the incubator at 25 &#176;C with a 12 h/12 h light/dark cycle.</li>
</ol>
2. Starvation Treatment for the Larvae
NOTE: As mentioned above, in the third instar larvae, there are few or no detectable lipid droplets in the oenocytes under normal feeding conditions, but numerous large lipid droplets can be induced in the oenocytes under stress conditions, such as starvation. To further verify this method, it is necessary to pretreat these larvae to induce lipid droplet biogenesis in oenocytes. Here, a starvation time course of 12 h, 24 h, and 36 h was chosen as the paradigm. In particular, a short period of starvation (e.g., 3 h) is sufficient to induce detectable lipid droplets in oenocytes. Starvation duration may vary according to specific experimental goals and settings.
<ol>
	<li>Make chambers for starvation and control treatments.
	<ol>
		<li>For starvation treatment chambers: place a filter paper of appropriate size in a 6 cm Petri dish and pipette 1 mL of PBS onto the filter paper.</li>
		<li>For control treatment chambers: place 5 mL of Bloomington standard cornmeal food in the Petri dish.</li>
	</ol>
	</li>
	<li>Use a spatula to gently dig up the top layer of food containing larvae that are still burrowing in the food and transfer them to a Petri dish filled with 5 mL of PBS. Gently stir the larvae in PBS to remove any food contamination from the larvae and make it as clean as possible.</li>
	<li>Use a small paintbrush to collect 40 third instar larvae of the same approximate size. Sort them randomly into a starvation or control chamber, with 20 larvae each.</li>
	<li>Place the chambers in the incubator at 25 &#176;C with 60% humidity and allow development for 12 h, 24 h, and 36 h of treatment. 
	NOTE: For the larvae in the starvation chamber, add 1 mL of PBS every 12 h to avoid larvae dehydration.</li>
</ol>
3. Dissection of Oenocytes
<ol>
	<li>Use a small paintbrush to pick larvae of the appropriate age (12 h, 24 h, or 36 h after treatment), then transfer them into a new Petri dish filled with 5 mL of ice-cold PBS to wash. 
	NOTE: Repeat step 2.2 when dealing with larvae in a control chamber to remove any food contamination.</li>
	<li>Fill a dissection plate with ice-cold PBS and use forceps to gently transfer larvae into the dissection plate. Put the dissection plate under a stereo microscope for the following dissection step. 
	NOTE: The ice-cold temperature will help slow movements of the larvae and facilitate dissection.</li>
	<li>Turn the larvae ventral side up and dorsal side down and gently hold in place using forceps. Secure the larvae to the dissection plate by placing a dissection pin though the pharynx at the anterior end and another pin through the spiracle at the posterior end. 
	NOTE: The dorsal side is most easily identified by presence of the dorsal trunks of the trachea.</li>
	<li>Use Vannas spring scissors to incise (longitudinally) through the epidermis from the anterior to the posterior end.</li>
	<li>Remove the internal tissue of the epidermis using forceps. 
	NOTE: Caution must be taken when removing the tracheal branches to avoid damage to the oenocytes, which are localized in the internal surface of epidermis.</li>
	<li>With forceps, retrieve the dissection pins and transfer the epidermis into a 1.5 mL microcentrifuge tube filled with PBS on ice.</li>
	<li>Continue to dissect other larvae following the procedure described above.</li>
</ol>
4. Lipid Droplet Staining
<ol>
	<li>Incubate the dissected epidermis in the fixation bufferfor 30 min at room temperature (RT) on a rotator. 
	NOTE: The fixation buffer contains 4% paraformaldehyde (PFA) in PBS.</li>
	<li>Remove the fixation buffer, followed by a quick wash. To perform a quick wash, add 1 mL of PBS at RT into the tube after the removal of PFA, gently resuspend the tissues, and discard the PBS. 
	CAUTION: The fixation buffer contains PFA, which is harmful to human health. It is important to properly dispose the fixation buffer as hazardous waste.</li>
	<li>Wash the samples 3x for 5 min each with PBS to wash out all possible PFA residue.</li>
	<li>Incubate the epidermis with BODIPY 493/503 (1 &#181;g/mL; see Table of Materials) for 30 min at RT on a rotator. 
	NOTE: From this step onwards, wrap the microcentrifuge tube with a piece of aluminum foil to protect samples from light and minimize the possible photo-bleaching.</li>
	<li>Remove the BODIPY 493/503 staining solution and wash the samples 3x for 10 min each with PBS to completely remove residual dyes.</li>
</ol>
5. Mounting and Imaging
<ol>
	<li>Place 6 &#181;L of mounting medium on a clean microscope slide. 
	NOTE: Mounting medium is used for longer detection time based on its antifade properties.</li>
	<li>Use forceps to pick up one epidermis and gently remove the residual PBS with a wipe.</li>
	<li>Place the epidermis in the mounting medium and adjust its orientation so that its internal surface containing the oenocytes is on the bottom and its external surface is on the top.</li>
	<li>Gently place a coverslip onto the epidermis. 
	NOTE: Remove any extra mounting medium leaking from under the coverslip with a wipe, if necessary. To facilitate imaging, gently push down on the coverslip with forceps so that when observing the slide through the microscope, the oenocyte cluster can be easily imaged in one single plane. Alternatively, it is practicable to cut the epidermis into two semi-epidermis through the middle line to avoid rolling-up of the whole epidermis when mounting the tissues.</li>
	<li>Apply clear nail polish around the edges of the coverslip to seal.</li>
	<li>Put the slides in a lightproof sample box and allow the nail polish to dry at RT, which may take 5-10 min.</li>
	<li>Proceed with microscopic analysis. Take images using a confocal microscope (magnification of 63x with optimized GFP or FITC filter settings, excitation = 488 nm, emission = 503 nm) to acquire clean and sensitive signals with minimized background.</li>
</ol>

<ol>
	<li>Liu, Z., Huang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+metabolism+in+Drosophila:+development+and+disease.">Lipid metabolism in Drosophila: development and disease.</a> Acta Biochimica et Biophysica Sinica (Shanghai). 45 (1), 44-50 (2013).</li><li>Cheng, Y., Chen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruit+fly+research+in+China.">Fruit fly research in China.</a> Journal of Genetics and Genomics. 45 (11), 583-592 (2018).</li><li>Warr, C. G., Shaw, K. H., Azim, A., Piper, M. D. W., Parsons, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+mouse+and+Drosophila+models+to+investigate+the+mechanistic+links+between+diet,+obesity,+type+II+diabetes,+and+cancer.">Using mouse and Drosophila models to investigate the mechanistic links between diet, obesity, type II diabetes, and cancer.</a> International Journal of Molecular Science. 19 (12), (2018).</li><li>Gutierrez, E., Wiggins, D., Fielding, B., Gould, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specialized+hepatocyte-like+cells+regulate+Drosophila+lipid+metabolism.">Specialized hepatocyte-like cells regulate Drosophila lipid metabolism.</a> Nature. 445 (7125), 275-280 (2007).</li><li>Makki, R., Cinnamon, E., Gould, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+and+functions+of+oenocytes.">The development and functions of oenocytes.</a> Annual Review of Entomology. 59, 405-425 (2014).</li><li>Chatterjee, D., 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=Control+of+metabolic+adaptation+to+fasting+by+dILP6-induced+insulin+signaling+in+Drosophila+oenocytes.">Control of metabolic adaptation to fasting by dILP6-induced insulin signaling in Drosophila oenocytes.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (50), 17959-17964 (2014).</li><li>Yan, Y., 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=HDAC6+suppresses+age-dependent+ectopic+fat+accumulation+by+maintaining+the+proteostasis+of+PLIN2+in+Drosophila.">HDAC6 suppresses age-dependent ectopic fat accumulation by maintaining the proteostasis of PLIN2 in Drosophila.</a> Developmental Cell. 43 (1), 99-111 (2017).</li><li>Yan, Y., 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=HDAC6+regulates+lipid+droplet+turnover+in+response+to+nutrient+deprivation+via+p62-mediated+selective+autophagy.">HDAC6 regulates lipid droplet turnover in response to nutrient deprivation via p62-mediated selective autophagy.</a> Journal of Genetics and Genomics. 46 (4), 221-229 (2019).</li><li>Farese, R. V., Walther, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+droplets+finally+get+a+little+R-E-S-P-E-C-T.">Lipid droplets finally get a little R-E-S-P-E-C-T.</a> Cell. 139 (5), 855-860 (2009).</li><li>Murphy, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+roles+of+intracellular+lipid+droplets:+from+archaea+to+mammals.">The dynamic roles of intracellular lipid droplets: from archaea to mammals.</a> Protoplasma. 249 (3), 541-585 (2012).</li><li>Tennessen, J. M., Barry, W. E., Cox, J., Thummel, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+studying+metabolism+in+Drosophila.">Methods for studying metabolism in Drosophila.</a> Methods. 68 (1), 105-115 (2014).</li><li>Fowler, S. D., Greenspan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Nile+red,+a+fluorescent+hydrophobic+probe,+for+the+detection+of+neutral+lipid+deposits+in+tissue+sections:+comparison+with+oil+red+O.">Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O.</a> Journal of Histochemistry &amp; Cytochemistry. 33 (8), 833-836 (1985).</li><li>Gocze, P. M., Freeman, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+underlying+the+variability+of+lipid+droplet+fluorescence+in+MA-10+Leydig+tumor+cells.">Factors underlying the variability of lipid droplet fluorescence in MA-10 Leydig tumor cells.</a> Cytometry. 17 (2), 151-158 (1994).</li><li>Fam, T. K., Klymchenko, A. S., Collot, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+fluorescent+probes+for+lipid+droplets.">Recent advances in fluorescent probes for lipid droplets.</a> Materials (Basel). 11 (9), (2018).</li><li>BDSC Standard Cornmeal Medium. Bloomington Drosophila Stock Center Available from: <a target="_blank" href="https://bdsc.indiana.edu/information/recipes/bloomfood.html">https://bdsc.indiana.edu/information/recipes/bloomfood.html</a> (2019)</li><li>Milon, A., 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=Do+estrogens+regulate+lipid+status+in+testicular+steroidogenic+Leydig+cell?.">Do estrogens regulate lipid status in testicular steroidogenic Leydig cell?.</a> Acta Histochemica. 121 (5), 611-618 (2019).</li><li>Farmer, B. C., Kluemper, J., Johnson, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apolipoprotein+E4+alters+astrocyte+fatty+acid+metabolism+and+lipid+droplet+formation.">Apolipoprotein E4 alters astrocyte fatty acid metabolism and lipid droplet formation.</a> Cells. 8 (2), (2019).</li></ol>

The authors have no conflicts of interest to disclose.

Among those outlined above, there are several critical steps in this protocol, with the egg laying time period being one of these. As a lipid-mobilizing tissue, the oenocyte is highly sensitive to nutrition status6,8. Prolonged egg-laying time periods may result in crowed larvae and increased food competition, leading to inaccurate results. The 1 h egg-laying time period used in this protocol allows the larvae to develop without nutrition competition. A much larg...

Lipids are essential for cell survival. In addition to their traditional role as integral components of cellular membrane systems, lipids also play crucial functions in energy supply and signaling transduction throughout the life cycles of individual animals1. Thus, lipid metabolism must conform to strict regulations to maintain physiological hemostasis in cells. It is known that dysregulation of lipid metabolism results in various diseases, such as diabetes and fatty liver. Despite the great importance of lipid metabolism in animal health, the mechanisms underlying lipid metabolism regulation remain largely unknown.
Drosophila has been extensively used for years since professor Thomas H. Morgan started to use them in studies involving genetics and other basic biological questions2. In the last few decades, emerging evidence has shown that Drosophila is an excellent model organism in the study of many lipid metabolism-associated diseases, such as obesity1,3. In particular, Drosophila shares highly conserved metabolic genes with humans and possess similar relevant tissues/organs and cell types for lipid metabolism.
For example, the fat body of Drosophila, which is responsible for triacylglyceride storage, functions analogous to human adipose tissue. Recently, a cluster of specialized hepatocyte-like cells (i.e., oenocytes), which have been reported to be a functional analog to the human liver, have been shown to be involved in fatty acid and hydrocarbon metabolism in fruit flies4,5. Similar to the case in mammal livers, oenocytes respond to starvation by activating lipid droplet formation in both larval and adult Drosophila, resulting in lipid droplet accumulation in oenocytes4,6,7,8. Anatomically, oenocytes are tightly attached to the basal internal surface of the lateral epidermis in clusters of approximately six cells per abdominal hemisegment, which makes it impracticable to isolate oenocyte clusters from the epidermis. Thus, oenocytes must be attached to the epidermis during dissection and staining.
Lipids are stored in the form of lipid droplets, which are organelles with single layer membranes in cells9. Lipid droplets exist in almost all cell types across different species10. Lipid droplet dynamics, including its size and number, change in response to environmental stressors. This is regarded as a reflection of metabolic status in response to stress, such as aging and starvation7,8. Therefore, it is of great importance to develop a feasible and reliable method to qualitatively determine lipid droplet dynamics in oenocytes during development and under stressful conditions. In particular, in the third instar larvae, oenocytes contain few or no detectable lipid droplets under fed conditions, but they do contain numerous large lipid droplets after nutrition deprivation4. To verify the effectiveness of this method, it is suggested to perform lipid droplet staining in the oenocytes under starved conditions.
Currently, several lipophilic dyes are available for lipid droplets staining, such as the nonfluorescent dyes Sudan Black and Oil Red O and fluorescent dyes Nile Red and BODIPY 493/50311. Sudan Black and Oil Red O are commonly used for tissue cholesteryl esters and triacylglycerols and can be easily detected by light microscopy. However, relatively high background staining and relatively low resolution are two limiting factors for its applications in qualitative analysis of lipid droplet dynamics. To overcome the limitations of nonfluorescent dyes, Nile Red and BODIPY 493/503 are utilized as ideal substitutes for lipid droplet staining. It has been reported that Nile Red can also detect some unesterified cholesterol, which makes BODIPY 493/503 a more specific dye for cellular lipid droplets, to some extent12,13,14.
Above all, to fulfill a need for rapid and sensitive analysis of lipid droplets in oenocytes, this protocol presents a feasible and highly reproducible method of fixative-based lipid droplet-specific staining using BODIPY 493/503 as the stain dye. In this report, oenocytes are dissected, and BODIPY 493/503 is used for lipid droplet staining in the oenocytes, in which lipid droplets are detected by confocal microscopy. The ease and affordability of this procedure make it ideal for modification and further use in other applications, such as flow cytometry.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>Corning</td>
			<td>430829</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>6 cm Petri dish</td>
			<td>Thermo Fisher</td>
			<td>150326</td>
			<td>6 cm</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td></td>
			<td></td>
			<td>For fly food</td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Protect smaple from light</td>
		</tr>
		<tr>
			<td>BODIPY 493/503</td>
			<td>Invitrogen</td>
			<td>D3922</td>
			<td>Lipid droplet staining dye</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica</td>
			<td>Leica TSC SP5</td>
			<td>Confocal imaging</td>
		</tr>
		<tr>
			<td>Corn syrup</td>
			<td></td>
			<td></td>
			<td>For fly food</td>
		</tr>
		<tr>
			<td>Cornmeal</td>
			<td></td>
			<td></td>
			<td>For fly food</td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>Citoglas</td>
			<td>10212424C</td>
			<td>20 &#215; 20 mm, 0.13-0.17 thick</td>
		</tr>
		<tr>
			<td>Dissection pin</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection plate</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Diameter: 11 cm</td>
		</tr>
		<tr>
			<td>Fixation buffer</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>4% Paraformaldehyde (PFA) in 1xPBS</td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>Dumont</td>
			<td>11252-30</td>
			<td>#5</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Jiangnan</td>
			<td>SPX-380</td>
			<td>For fly culture</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td>1.5 ml</td>
		</tr>
		<tr>
			<td>Microscopy slide</td>
			<td>Citoglas</td>
			<td>10127105P-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium</td>
			<td>VECTASHIELDAntifade Mounting Medium</td>
			<td>H-1000</td>
			<td>Antifade mounting medium</td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>PanEra</td>
			<td>AAPR419</td>
			<td>Seal the coverslip</td>
		</tr>
		<tr>
			<td>Paintbrush</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>1xPBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,1.8 mM KH2PO4, pH 7.4)</td>
		</tr>
		<tr>
			<td>Rotator</td>
			<td>Kylin-Bell Lab Instruments</td>
			<td>WH-986</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td>Smartdata Medical</td>
			<td>SR81</td>
			<td>Vannas spring scissor</td>
		</tr>
		<tr>
			<td>Soy flour</td>
			<td></td>
			<td></td>
			<td>For fly food</td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard cornmeal food</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Accoding to Bloomington standard cornmeal food recipe</td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Leica</td>
			<td>Leica S6E</td>
			<td>For tissue dissection</td>
		</tr>
		<tr>
			<td>Wipe paper</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast</td>
			<td></td>
			<td></td>
			<td>For fly food</td>
		</tr>
		<tr>
			<td>yw</td>
			<td>Kept as lab stock</td>
			<td>N/A</td>
			<td>Drosophila</td>
		</tr>
	</tbody>
</table>

dissection and lipid droplet staining of oenocytes in drosophila larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and diseases, such as obesity and fatty liver. Usually, lipids are stored in lipid droplets, which are the multifunctional lipid storage organelles in cells. Lipid droplets vary in size and number in different tissues and under different conditions. It has been reported that lipid droplets are tightly controlled through regulation of its biogenesis and degradation. In Drosophila melanogaster, the oenocyte is an important tissue for lipid metabolism and has been recently identified as a human liver analogue regarding lipid mobilization in response to stress. However, the mechanisms underlying the regulation of lipid droplet metabolism in oenocytes remain elusive. To solve this problem, it is of utmost importance to develop a reliable and sensitive method to directly visualize lipid droplet dynamic changes in oenocytes during development and under stressful conditions. Taking advantage of the lipophilic BODIPY 493/503, a lipid droplet-specific fluorescent dye, described here is a detailed protocol for the dissection and subsequent lipid droplet staining in the oenocytes of Drosophila larvae in response to starvation. This allows for qualitative analysis of lipid droplet dynamics under various conditions by confocal microscopy. Furthermore, this rapid and highly reproducible method can also be used in genetic screens for indentifying novel genetic factors involving lipid droplet metabolism in oenocytes and other tissues.

Successful execution of this procedure should result in clear lipid droplets staining that reveals the number and size of lipid droplets in the oenocytes. Figure 1A,A&#39;,A&#39;&#39; shows that there are few detectable lipid droplets (green dots) in the oenocytes of normal feeding larvae during different developmental stages. Figure 1B,B&#39;,B&#39;&#39; shows increased lipid droplets (green dot...

Watch this Scientific Journal Video about Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae at JoVE.com

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...

dissection-lipid-droplet-staining-oenocytes-drosophila

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11.5K Views.  Guangzhou Medical University. Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

Video: Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

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Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Dissection z larw CNS na Drosophila melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

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 ...

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...