Name: Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos (Video) | JoVE Journal
Uploaded: 2010-12-11T12:00:00.000+00:00
Duration: 9 min 8 s
Description: Learn to visualize the embryonic nervous system in whole-mount Drosophila embryos using immunofluorescence microscopy. This protocol enables researchers to study neuronal structures and genetic influences on development.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

playerTrialDesktop

SG is supported by funds from the State University of New York at Buffalo and from John R. Oishei Foundation.

1. Preparation of Reagents
<ol>
	<li>Prepare the PEMFA Buffer using 100mM Pipes, 2mM EGTA, and 1 mM MgSO4. Adjust the pH of the buffer to 7.0. The PEMFA buffer is filter sterilized and stored at 4°C.</li>
	<li>Prepare PBT by adding 1ml of Triton X-100 to 100ml 1x PBS (Phosphate Buffered Saline) (pH 7.0). Store at 4°C.</li>
	<li>Prepare 5% BSA (bovine serum albumin) with 0.01% NaAcide in 1x PBS (pH 7.0).</li>
	<li>Prepare the saline solution using 0.7% NaCl and 0.03% Triton X-100 in deionized water. </li>
	<li>Prepare the fixative by combining 4 parts PEMFA buffer with 1 part 37% Formaldehyde.</li>
</ol>
2. Collection of Embryos
<ol>
	<li>Prepare a cage for the Drosophila strain of interest by placing &gt;100 flies in a plastic bottle containing an adaptor with agar feeding plate.</li>
	<li>Leave the cage of flies in the dark at room temperature for 72 hours, changing plates every 8-12 hours, so that the flies get acclimated to the cage and do not hold their eggs.</li>
	<li>For the experiment collect embryos for 12 hours. Remove the Petri dish from the container and replace it. Take the removed Petri dish and wash the embryos off the dish using the saline solution and a clean paint brush.</li>
	<li>Collect the embryos in a fine sieve. Rinse with saline to remove any excess yeast. </li>
	<li>Using a spatula gently pick up the embryos and place them in a small glass vial with a cap. Add about 1ml of saline solution.</li>
</ol>
3. Removal of the Chorion and Vitelline Membranes and Fixation
<ol>
	<li>Following the collection of embryos, prepare the Heptane-Fix solution by combining 50% heptane and 50% fixative. The solution will be layered with heptane on top and the fixative on the bottom.</li>
	<li>In the glass vial wash the embryos by incubating them in the saline solution for 5 minutes. Remove excess saline using a Pasteur pipette. </li>
	<li>Rinse the embryos with deionized water. </li>
	<li>Dechorionate the embryos by incubating them in 50% bleach for 3 minutes. </li>
	<li>Rinse the embryos thoroughly with deionized water. </li>
	<li>Fix the embryos by incubating them for 30 minutes in the Heptane-Fix solution with gentle shaking.</li>
	<li>Using a Pasteur pipette replace the fixative (bottom layer) with methanol leaving the heptane in the tube. Shake vigorously to remove the vitelline membrane. The devitellinized embryos will sink to the bottom of the tube. </li>
	<li>Collect the embryos in a new glass vial using a Pasteur pipette. Rinse the embryos five times with methanol, each time incubate the embryos for 5 minutes.</li>
</ol>
4. Antibody Staining, Mounting, and Visualization
<ol>
	<li>Rehydrate the embryos in a mixture of 50% PBT solution and 50% methanol for 5 minutes. Then rehydrate in 100% PBT solution for 5 minutes. </li>
	<li>Block the embryos by incubating them in 5% BSA with 0.01% NaAcide solution for 1 hour at 4°C. </li>
	<li>Embryos are now ready for antibody staining. Prepare the primary antibody by diluting it to the appropriate concentration using 5%BSA with 0.01% NaAcide solution. We use an antibody against the cysteine string protein (CSP), a vesicular synaptic protein and an anti-HRP or horse radish peroxidase antibody, which recognizes a neuronal specific-carbohydrate moiety present in the surface glycoproteins and labels all neurons.</li>
	<li>Incubate the embryos overnight at 4°C in the primary antibody solution. </li>
	<li>The following day, wash the embryos 10 times with PBT over the course of an hour. </li>
	<li>Prepare the secondary antibody solution by diluting the appropriate concentration. We use Alexa secondary antibodies at a concentration of 1:200.</li>
	<li>Incubate the embryos for two hours in 4°C in the secondary antibody solution. </li>
	<li>Wash the embryos 10 times with PBT over the course of an hour. </li>
	<li>Incubate the embryos overnight in Vectashield Mounting Medium.</li>
	<li> Mount the embryos on a glass slide and seal the cover slip with nail polish. Visualize embryos using a fluorescence microscope.</li>
</ol>
5. Representative Results
<img src="/files/ftp_upload/2150/2150fig1a.jpg" alt="Confocal microscope image of fluorescent cells, scale bars showing magnification for cellular analysis." data-altupdatedat="1788235205000" data-alt="Figure 1"> CSP <img src="/files/ftp_upload/2150/2150fig1b.jpg" alt="Confocal microscopy image; spinal structure; fluorescence staining; cellular visualization." data-altupdatedat="1788235205000" data-alt="Figure 1"> HRP-FITC <img src="/files/ftp_upload/2150/2150fig1c.jpg" alt="Confocal microscopy image, cell structure visualization; fluorescent proteins highlight cellular morphology." data-altupdatedat="1788235205000" data-alt="Figure 1"> Merged Figure 1: A Representative images showing the embryonic CNS at stage 16-17, using antibodies against CSP (red) and HRP (green). Note the distinct staining of the commissure.
<img src="/files/ftp_upload/2150/2150fig2a.jpg" alt="Confocal microscopy image; nerve fibers; fluorescence labeling; cellular structure visualization." data-altupdatedat="1788235205000" data-alt="Figure 2"> CSP <img src="/files/ftp_upload/2150/2150fig2b.jpg" alt="Fluorescence microscopy image showing nerve fibers in a biological tissue sample, 20 μm scale." data-altupdatedat="1788235205000" data-alt="Figure 2"> HRP-FITC <img src="/files/ftp_upload/2150/2150fig2c.jpg" alt="Confocal microscopy image; neuron structure visualization; fluorescent labeling, magnification scale." data-altupdatedat="1788235205000" data-alt="Figure 2"> Merged Figure 2: A representative images showing the embryonic PNS at stage 16-17 using antibodies against CSP (red) and HRP (green). Note the repeated patterns of peripheral neurons.

<ol><li>Bodmer, R., Barbel, S., Sheperd, S., Jack, J. W., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transformation+of+sensory+organs+by+mutations+of+the+cut+locus+of+D.+melanogaster%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Transformation of sensory organs by mutations of the cut locus of D. melanogaster.</a> Cell. 51, 293-307 (1987).</li>
<li>Bodmer, R., Carretto, R., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurogenesis+of+the+peripheral+nervous+system+in+Drosophila+embryos%3A+DNA+replication+patterns+and+cell+lineages%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Neurogenesis of the peripheral nervous system in Drosophila embryos: DNA replication patterns and cell lineages.</a> Neuron. 3, 21-32 (1989).</li>
<li>Campos-Ortega, J. A., Hartenstein, V. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aCampos-Ortega%2C+JA+%22The+Embryonic+Development+of+Drosophila+melanogaster%22">The Embryonic Development of Drosophila melanogaster</a>. , Springer-Verlag. New York. (1985).</li>
<li>Carroll, S. B., Scott, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Localization+of+the+fushi+tarazu+protein+during+Drosophila+embryogenesis%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Localization of the fushi tarazu protein during Drosophila embryogenesis.</a> Cell. 43, 47-57 (1985).</li>
<li>Dambly-Chaudière, C., Ghysen, A., Jan, Y. N., Jan, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Muscle+connections+between+imaginal+discs+in+Drosophila%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Muscle connections between imaginal discs in Drosophila.</a> Developmental Biology. 113, 288-294 (1986).</li>
</ol>

No conflicts of interest declared.

The Drosophila embryo is a powerful system for investigating the cellular and molecular changes during neuronal development. In this protocol we have demonstrated how to prepare whole mount embryos for immunohistochemistry. We have adapted this protocol from the protocol described in Carroll and Scott, 1985. This protocol can also be adapted to test other neuronal antibodies, but optimization of antibodies should be done. Further, developmental defects of the embryonic PNS and CNS can be easily visualized by com...

<table><tbody><tr><td>37% formaldehyde</td><td>Fisher Scientific</td><td>BP531-25</td><td></td></tr><tr><td>CSP</td><td>Developmental Studies Hybridoma Bank</td><td></td><td></td></tr><tr><td>HRP-FITC</td><td>Jackson ImmunoResearch</td><td>323-095-021</td><td></td></tr><tr><td>Alexa Fluor 568 Goat Anti-Mouse IgG</td><td>Invitrogen</td><td>A-11004</td><td></td></tr><tr><td>Vectashield Mounting Medium</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr></tbody></table>

visualization of the embryonic nervous system in whole-mount drosophila embryos

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

The overall goal of this procedure is to prepare oph and melanogaster embryos for whole mount visualization of the nervous system. This is accomplished by first collecting embryos from the fly strain of interest. The second step of the procedure is to remove the corium with bleach.The third step of the procedure is to apply heptane and methanol to remove the vilin membrane. The final step of the procedure is to apply antibodies to the embryos. Ultimately, results can be obtained that show the structure of the drosophila embryo nervous system through immunofluorescence microscopy.This method can help answer key questions in the field of neuroscience, such as what role mutations can play in the developing nervous system. Generally, individuals new to this method will struggle because they may accidentally pipe out embryos from the vial. Begin this protocol by preparing the reagents that will be needed, including P-E-M-F-A, buffer PBC 5%BSA with 0.01%sodium azide saline solution and formaldehyde fixative.Prepare a cage for the drosophila strain of interest by knocking the flies out with carbon dioxide and then transferring at least 100 flies to a plastic bottle containing an adapter w

visualization-embryonic-nervous-system-whole-mount-drosophila

Research

JoVE Journal

Neuroscience

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

Upright Imaging of Drosophila Embryos

Source: Jeong, S. Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila. J. Vis. Exp. (2017)This video demonstrates the visualization of axonal projections in Drosophila embryos. Immunolabeling of motor neurons, precise dissection to expose neuronal structures, and differential interference contrast microscopy collectively reveal detailed neuronal architecture, emphasizing the axonal projection patterns.

Visualization of Axonal Projection Patterns of Embryonic Motor Neurons in Drosophila

Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae.

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Live Imaging of Drosophila Larval Neuroblasts

We describe a protocol for dissection, fixation, and immunostaining of steroidogenic organs in Drosophila larvae and adult females to study steroid hormone biosynthesis and its regulatory mechanism. In addition to steroidogenic organs, we visualize the innervation of steroidogenic organs as well as steroidogenic target cells such as germline stem cells.

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

We describe a method for retrograde tracing of the Drosophila embryonic motor neurons using lipophilic fluorescent dyes.

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

This protocol describes three steps to prepare larval and adult Drosophila optic lobes for imaging: 1) brain dissections, 2) immunohistochemistry and 3) mounting. Emphasis is placed on step 3, as distinct mounting orientations are required to visualize specific optic lobe...

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

Here, we provide a dissection protocol required to live-image the late embryonic Drosophila male gonad. This protocol will permit observation of dynamic cellular processes under normal conditions or after transgenic or pharmacological manipulation.

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Learn to visualize the embryonic nervous system in whole-mount Drosophila embryos using immunofluorescence microscopy. This protocol enables researchers to study neuronal structures and genetic influences on development.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

Explore More Videos