Name: dissection and immunostaining of larval salivary glands from anopheles gambiae mosquitoes
Uploaded: 2021-09-30T14:45:00
Description: Watch this Scientific Journal Video about Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes at JoVE.com

We would like to thank the Johns Hopkins Malaria Research Institute for access to and rearing of An. gambiae larvae.

1. Preparation of solutions and tools
<ol>
	<li>Preparation of dissection solution
	<ol>
		<li>To prepare dissecting solution, add 2.5 mL of 100% ethanol to 7.5 mL of distilled H2O in a 15 plastic tube. Invert the tube 3 times to mix. 
		NOTE: This solution can be stored at room temperature for several weeks.</li>
	</ol>
	</li>
	<li>Preparation of 10x phosphate-buffered saline (PBS) stock
	<ol>
		<li>To prepare 10x PBS stock, add 17.8 g Na2HPO4&#8226; 2H2</sub...

<ol>
	<li>World malaria report 2020: 20 years of global progress and challenges. World Health Organization Available from: <a target="_blank" href="https://apps.who.int/iris/handle/10665/337660">https://apps.who.int/iris/handle/10665/337660</a> (2020)</li><li>Feachem, R. G. 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=Malaria+eradication+within+a+generation:+ambitious,+achievable,+and+necessary.">Malaria eradication within a generation: ambitious, achievable, and necessary.</a> Lancet. 394 (10203), 1056-1112 (2019).</li><li>Adolfi, 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=Efficient+population+modification+gene-drive+rescue+system+in+the+malaria+mosquito+Anopheles+stephensi.">Efficient population modification gene-drive rescue system in the malaria mosquito Anopheles stephensi.</a> Nature Communications. 11, 5553 (2020).</li><li>Kyrou, K., 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=A+CRISPR-Cas9+gene+drive+targeting+doublesex+causes+complete+population+suppression+in+caged+Anopheles+gambiae+mosquitoes.">A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes.</a> Nature Biotechnology. 36 (11), 1062-1066 (2018).</li><li>Rostami, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9+gene+drive+technology+to+control+transmission+of+vector-borne+parasitic+infections.">CRISPR/Cas9 gene drive technology to control transmission of vector-borne parasitic infections.</a> Parasite Immunology. 42 (9), 12762 (2020).</li><li>Bhatt, 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=Coverage+and+system+efficiencies+of+insecticide-treated+nets+in+Africa+from+2000+to+2017.">Coverage and system efficiencies of insecticide-treated nets in Africa from 2000 to 2017.</a> Elife. 4, 09672 (2015).</li><li>Mellink, J. 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S., Sumitani, M., Kasashima, K., Sezutsu, H., Matsuoka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+malaria+infection+in+transgenic+Anopheline+mosquitoes+lacking+salivary+gland+cells.">Inhibition of malaria infection in transgenic Anopheline mosquitoes lacking salivary gland cells.</a> PLoS Pathogens. 12 (9), 1005872 (2016).</li><li>Rishikesh, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+and+development+of+the+salivary+glands+and+their+chromosomes+in+the+larvae+of+Anopheles+stephensi+sensu+stricto.">Morphology and development of the salivary glands and their chromosomes in the larvae of Anopheles stephensi sensu stricto.</a> Bulletin of the World Health Organization. 20 (1), 47-61 (1959).</li><li>Jensen, D. V., Jones, J. C. <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+of+the+salivary+glands+in+Anopheles+albimanus+Wiedemann+(Diptera,+Culicidae).">The development of the salivary glands in Anopheles albimanus Wiedemann (Diptera, Culicidae).</a> Annals of the Entomological Society of America. 50 (5), 40824 (1957).</li><li>Chiu, M., Trigg, B., Taracena, M., Wells, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+cellular+morphologies+during+lumen+maturation+in+Anopheles+gambiae+larval+salivary+glands.">Diverse cellular morphologies during lumen maturation in Anopheles gambiae larval salivary glands.</a> Insect Molecular Biology. 30 (2), 210-230 (2021).</li><li>MR4. Methods in Anopheles research. Center for Disease Control Available from: <a target="_blank" href="https://www.beiresources.org/Portals/2/">https://www.beiresources.org/Portals/2/</a> (2015)</li><li>Wells, M. B., Andrew, 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=Salivary+gland+cellular+architecture+in+the+Asian+malaria+vector+mosquito+Anopheles+stephensi.">Salivary gland cellular architecture in the Asian malaria vector mosquito Anopheles stephensi.</a> Parasites &amp; Vectors. 8, 617 (2015).</li><li>Wells, M. B., Villamor, J., Andrew, 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=Salivary+gland+maturation+and+duct+formation+in+the+African+malaria+mosquito+Anopheles+gambiae.">Salivary gland maturation and duct formation in the African malaria mosquito Anopheles gambiae.</a> Scientific Reports. 7 (1), 601 (2017).</li><li>Wells, M. B., Andrew, 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=Anopheles+salivary+gland+architecture+shapes+plasmodium+sporozoite+availability+for+transmission.">Anopheles salivary gland architecture shapes plasmodium sporozoite availability for transmission.</a> mBio. 10 (4), 01238 (2019).</li><li>Clements, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The physiology of mosquitoes. , (1963).</li><li>Imms, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+larval+and+pupal+stages+of+Anopheles+maculipennis,+meigen.">On the larval and pupal stages of Anopheles maculipennis, meigen.</a> Parasitology. 1 (2), 103-133 (1908).</li><li>Favia, G., 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=Bacteria+of+the+genus+Asaia+stably+associate+with+Anopheles+stephensi,+an+Asian+malarial+mosquito+vector.">Bacteria of the genus Asaia stably associate with Anopheles stephensi, an Asian malarial mosquito vector.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (21), 9047-9051 (2007).</li><li>Neira Oviedo, M., 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=The+salivary+transcriptome+of+Anopheles+gambiae+(Diptera:+Culicidae)+larvae:+A+microarray-based+analysis.">The salivary transcriptome of Anopheles gambiae (Diptera: Culicidae) larvae: A microarray-based analysis.</a> Insect Biochemistry and Molecular Biology. 39 (5-6), 382-394 (2009).</li><li>Linser, P. J., Smith, K. E., Seron, T. J., Neira Oviedo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbonic+anhydrases+and+anion+transport+in+mosquito+midgut+pH+regulation.">Carbonic anhydrases and anion transport in mosquito midgut pH regulation.</a> Journal of Experimental Biology. 212 (11), 1662-1671 (2009).</li><li>Linser, P. J., 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=Slc4-like+anion+transporters+of+the+larval+mosquito+alimentary+canal.">Slc4-like anion transporters of the larval mosquito alimentary canal.</a> Journal of Insect Physiology. 58 (4), 551-562 (2012).</li></ol>

The protocol described herein was adapted from a Drosophila SG dissection protocol and an adult mosquito dissection protocol14,15,16. However, most markers did not penetrate the basement membrane (data not shown) when using the adult dissection and SG staining methods. Adaptations of the adult protocol included dissecting the glands in a 25% EtOH solution, washing the glands with a combination of MeOH and glacial acetic acid, an...

Malaria is a major public health threat causing almost 230 million infections and an estimated 409,000 deaths in 20191. The majority of deaths are in sub-Saharan Africa and are caused by the parasite Plasmodium falciparum, whose insect vector is Anopheles gambiae, the subject of this video demonstration. Although the numbers indicate a significant drop in annual death rate since the turn of the century (&#62;300,000 fewer annual deaths), the promising decreases in disease rates observed from 2000 to 2015 are tapering, suggesting the need for new approaches to limiting disease transmission2. Among promising additional strategies for controlling and possibly eliminating malaria is targeting mosquito vector capacity using CRISPR/Cas9-based gene-editing and gene-drive3,4,5. Indeed, it is the targeting of the mosquito vector (through the expanded use of long-lasting insecticide-treated bed nets) that has had the greatest impact on reducing disease transmission6.
Female mosquitoes acquire Plasmodium gametocytes from an infected human during a blood meal. Following fertilization, maturation, midgut epithelium traversal, population expansion, and hemocoel navigation in their obligate mosquito hosts, hundreds to tens of thousands of Plasmodium sporozoites invade the mosquito SGs and fill the secretory cavities of the constituent secretory cells. Once inside the secretory cavities, the parasites have direct access to the salivary duct and are thus poised for transmission to a new vertebrate host upon the next blood meal. Because SGs are critical for the transmission of malaria-causing sporozoites to their human hosts, and laboratory studies suggest that SGs are not essential for blood-feeding, mosquito survival, or fecundity7,8,9, they represent an ideal target for transmission-blocking measures. Adult mosquito SGs form as an elaboration of &#34;duct bud&#34; remnants in the larval SGs that persist beyond early pupal SG histolysis10, making the larval SG an ideal target for interventions to limit adult-stage disease transmission.
Characterizing the larval stage of SG development can help develop not only a better understanding of its morphology and functional adaptations but can also aid in assessing new interventions that target this organ through gene editing of key SG regulators. Because all previous studies of larval salivary gland architecture predate immunostaining and modern imaging techniques10,11, we have developed a protocol for isolating and staining salivary glands with a variety of antibodies and cell markers12. This video demonstrates this approach to the extraction, fixation, and staining of larval SGs from Anopheles gambiae L4 larvae for confocal imaging.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;KH2PO4</td>
			<td>Millipore Sigma</td>
			<td>P9791</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Na2HPO4 &#8226; 2H2O</td>
			<td>Millipore Sigma</td>
			<td>71643</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;NaCl</td>
			<td>Millipore Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Millipore Sigma</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr>
			<td>Brush with soft bristles</td>
			<td>Amazon (SN NJDF) Detail Paint Brush Set</td>
			<td>B08LH63D89</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slips (22 x 50 mm)</td>
			<td>VWR</td>
			<td>48393-195</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (DNA)</td>
			<td>ThermoFisher Scientific</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol 200 proof</td>
			<td>Millipore Sigma</td>
			<td>EX0276</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilson Pipetman P200 Pipette</td>
			<td>Gilson</td>
			<td>P200</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>Sigma Aldrich</td>
			<td>695092</td>
			<td></td>
		</tr>
		<tr>
			<td>Jewelers forceps, Dumont No. 5</td>
			<td>Millipore Sigma</td>
			<td>F6521</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Millipore Sigma</td>
			<td>58221</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Millipore Sigma</td>
			<td>1414209</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>Amazon (Sally Hansen)</td>
			<td>B08148YH9M</td>
			<td></td>
		</tr>
		<tr>
			<td>Nile Red (lipid)</td>
			<td>ThermoFisher Scientific</td>
			<td>N1142</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper towels/wipes</td>
			<td>ULINE</td>
			<td>S-7128</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri plate (to make putty plate)</td>
			<td>ThermoFisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips</td>
			<td>Gilson</td>
			<td>Tips E200ST</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Transfer Pipette</td>
			<td>Fisher Scientific</td>
			<td>S304671</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary antibodies (e.g., Crb, Rab11)</td>
			<td>Developmental Studies Hybridoma Bank (DSHB); Andrew Lab</td>
			<td>Mouse anti-Crb (Cq4) or Rabbit anti-Rab11</td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary antibodies with fluorescent tags (e.g., Alexa Fluor 488 Goat-anti Rabbit)</td>
			<td>ThermoFisher Scientific</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone resin and curing agent for putty plate</td>
			<td>Dow Chemicals - Ximeter Silicone</td>
			<td>PMX-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides, frosted on one end for labelling</td>
			<td>VWR&#160; 20 X 50 mm</td>
			<td>48393-195</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheat Germ Agglutinin</td>
			<td>ThermoFisher Scientific</td>
			<td>W834</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection and immunostaining of larval salivary glands from anopheles gambiae mosquitoes

Mosquito salivary glands (SGs) are a requisite gateway organ for the transmission of insect-borne pathogens. Disease-causing agents, including viruses and the Plasmodium parasites that cause malaria, accumulate in the secretory cavities of SG cells. Here, they are poised for transmission to their vertebrate hosts during a subsequent blood meal. As adult glands form as an elaboration of larval SG duct bud remnants that persist beyond early pupal SG histolysis, the larval SG is an ideal target for interventions that limit disease transmission. Understanding larval SG development can help develop a better understanding of its morphology and functional adaptations and aid in the assessment of new interventions that target this organ. This video protocol demonstrates an efficient technique for isolating, fixing, and staining larval SGs from Anopheles gambiae mosquitoes. Glands dissected from larvae in a 25% ethanol solution are fixed in a methanol-glacial acetic acid mixture, followed by a cold acetone wash. After a few rinses in phosphate-buffered saline (PBS), SGs can be stained with a broad array of marker dyes and/or antisera against SG-expressed proteins. This method for larval SG isolation could also be used to collect tissue for in situ hybridization analysis, other transcriptomic applications, and proteomic studies.

Salivary glands are relatively easy to dissect from all stage 4 larvae. Male and female larvae can be distinguished at the late L4 larval stage by a red stripe along the dorsal thorax of females but not males (Figure 2). We also observe that antennal morphology is much more elaborate in male than in female L4 larvae (Figure 2), similar to the differences observed in this structure in adult mosquitoes. Along with the considerable overall growth during the L4 stag...

Watch this Scientific Journal Video about Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes at JoVE.com

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes

The adult mosquito salivary gland (SG) is required for the transmission of all mosquito-borne pathogens to their human hosts, including viruses and parasites. This video demonstrates efficient isolation of the SGs from the larval (L4) stage Anopheles gambiae mosquitoes and preparation of the L4 SGs for further analysis.

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes

Mosquito salivary glands (SGs) are a requisite gateway organ for the transmission of insect-borne pathogens. Disease-causing agents, including viruses and ...

The salivary glands of insects, are required for transmitting many human diseases. So a better understanding, of salary gland biology should help us, add to existing strategies for disease prevention. This technique will allow us, to quickly ask if mutations in the candidate regulators, affect the morphology, of the mosquito larval salivary glands, and potentially affect the transmission, of malarial parasites.The first few times you work with the mosquito larvae, they are challenging to grasp as they move quickly. The more you practice, the better you get. Getting started with the procedure, will be Marisol Luchetti, an undergraduate research assistant for my laboratory.To start the experiment, place a potty plate on the stage of a dissecting microscope, and transfer 10 mosquito L4 larvae onto the potty plate. Then place a drop of the dissection solution, onto the potty plate separate from the larvae. Use forceps to place one L4 larva, into the dissection solution drop of 25%ethanol.By holding a number five forceps in each hand, grasp the larvae with forceps just below the head, with the dominant hand, and grip the head of the larva with the non-dominant hand, then gently pull the head with minimal constant force, to detach from the rest of the body, while the glands remain attached to the head. After discarding the body portion of the larva carcass, collect the head or salivary glands, into one milliliter of PBS, in a 1.5 milliliter micro centrifuge tube, and wait for the salivary glands, to sink to the bottom of the buffer. On the next day, drain the solution to fixate the tissues, with one milliliter of cold 100%acetone for 90 seconds.After removing the acetone, gently rinse the tissues three times, with one milliliter of PBS. For immunostaining, add a primary antibody such as Rab 11, at the appropriate dilution, in 200 microliters of the total volume of PBS. Swirl the tube contents gently with a pipette tip.Incubate the primary antibodies overnight at four degrees, followed by washing three times in one milliliter of PBS. Then add the secondary antibody, Alexa Fluor 488 Goat anti-Rabbit, at the appropriate dilution, in 200 microliters of total volume. After mixing the contents with a pipette tip, incubate the tissues with dyes, such as Nile red, and Hoechst into 200 microliters of PBS, at room temperature for 60 minutes, followed by washing three times with PBS.Prepare a microscope slide, by adding 200 microliters of 100%glycerol with a pipette, and transferring up to 20 stained heads, with the attached glands and internal structures, per microscope slide using a soft brush. Spread the head samples out, to evenly distribute them along the glass slide. Viewing under a dissecting microscope, separate the larval head from the glands, using two pairs of forceps, by carefully pulling the two tissues in opposite directions.When all the samples are done, gently place a 1.5 millimeter thick cover slip, on top of the slide, avoiding the formation of air bubbles, and then seal the slide with clear nail Polish. The salivary glands are relatively easy to dissect, from stage four larvae. Male and female larvae can be distinguished, at the late L4 larval stage by a red stripe, along the dorsal thorax of females.The antennal morphology is also more elaborate, in males than in female L4 larvae as can be seen, with the white arrows pointing out, the female antennae on the left image, and the male antennae on the right. The salivary glands isolated from early L4 stage larvae, stained with Hoechst reveal the proximal and distal lobes, separated by a narrow constriction. The forming lumen was examined, in the immuno stained distal salivary gland, of a mid to late L4 larva.The apical domains of the secretory cells, surrounding the forming lumen had intense Nile red staining, suggestive of microvilli like structures. The Rab 11 staining was observed close, to the apical surface. There's only one step with the time, and that's the 90 second incubation, of the tissues in cold acetone.This protocol was only recently developed, but we expect it will be helpful, to anyone working on the mosquito salivary gland. We fully expect to use it often.

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