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 y...

<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 ...

This method of an oenocyte dissection and lipid droplet staining can be used to realize the appearance of lipid droplet in drosophila larvae oenocyte under normal and stressed conditions. This method provide a useful tool for studying lipid metabolism not only in insects but also in mammals with only a few minor modifications. To induce egg laying, place 150 virgin female flies and 75 males of the desired genotypes in an egg-laying bottle and place the bottle under a light-proof box in a 25 degrees Celsius incubator with 60%humidity.After one hour, transfer the flies to a new bottle and let the flies lay eggs in the new bottle for one hour before removing the adults. Then, allow the eggs to develop into the third instar larvae for 84 hours at 25 degrees Celcius with a 12-hour light-dark cycle. Before initiating the starvation treatment, place an appropriately sized piece of filter paper into a six centimeter Petri dish and add one milliliter of PBS onto the paper to create a starvation chamber.To make a control treatment chamber, place five milliliters of Bloomington standard cornmeal food into a six centimeter Petri dish. When the chambers are ready, use a small paint brush to collect instar larvae of the same approximate size from the egg-laying bottle. And 20 larvae randomly into each starvation and the control chamber.Then place the chambers in the incubator for 12, 24 or 36 hours of development and/or starvation. At the end of the treatment period, use forceps to gently transfer larvae from each chamber into a dissection plate containing ice cold PBS and place the plate under a stereo microscope. Orient the first larva ventral side up and use forceps to gently hold the larva in place.Place one pin through the pharynx and another pin through the spiracle to secure the larva to the plate. Use Vannas spring scissors to make a longitudinal incision through the epidermis from the anterior to the posterior end of the animal. Use forceps to remove the internal tissue of the epidermis, taking care to avoid damage to the oenocytes on the internal surface of the epidermis.Then use forceps to retrieve the dissection pins and transfer the epidermis to a 1.5 milliliter microcentrifuge tube of PBS on ice. For lipid droplet staining, incubate the dissected epidermis in fixation buffer for 30 minutes at room temperature on a rotator, followed by a quick resuspension in one milliliter of PBS. Then wash the samples with three five-minute washes in one milliliter of PBS per wash to remove any residual fixative.After the last wash, incubate the epidermal samples with an appropriate lipophilic probe for 30 minutes at room temperature on a rotator. During the incubation, wrap the sample tubes in foil to protect the tissues from light and wash the samples three times in one milliliter of PBS for 10 minutes per wash. To image the oenocytes, transfer each epidermis sample into six microliters of mounting medium on a clean microscope slide, adjust the orientation of the tissue, so that the internal surface containing the oenocytes is touching the bottom of the slide.Gently place a coverslip onto the epidermis and seal the edges with clear nail polish. When the polish has dried, image the samples on a confocal microscope, put a 63 X magnification with the appropriate excitation and emission wavelengths. There are a few detectable lipid droplets within the oenocytes of normal feeding larvae at different developmental stages.As observed in these representative images however, the lipid droplets increase in number within the oenocytes in response to 12, 24, and 36-hour periods of starvation. This method provides and easy and a feasible protocol for researchers in relevant research fields to investigate whether genetic or environmental manipulations cause lipid droplet changes in cells.

dissection-lipid-droplet-staining-oenocytes-drosophila

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Biology

11.2K vues.  Guangzhou Medical University. Cette méthode d’une dissection d’œnocyte et d’une coloration des gouttelettes lipidiques peut être utilisée pour réaliser l’apparition d’une gouttelette lipidique dans l’œnocyte des larves de drosophiles dans des conditions normales et stressées. Cette méthode fournit un outil utile pour étudier le métabolisme des lipides non seulement chez les insectes, mais aussi chez les mammifères avec seulement quelques modifications mineures. Pour induire la ponte, placez 150 mouc...