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.

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5 minutes

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 with an apple juice agar feeding plate. Place the cage of flies in the dark at room temperature for 72 hours. Change the apple juice agar feeding plates every eight to 12 hours so that the flies get acclimated to the cage and do not hold their eggs for the experiment.After the 72 hour acclimation period, collect embryos 12 hours from the last change in apple juice agar feeding plate. Remove the apple juice agar feeding plate from the laying cage and replace it using saline solution. Rinse the apple juice agar feeding plate.Collect the embryos in a fine sieve. Use a clean paint brush to dislodge any embryos that remain stuck to the plate. Rinse the embryos in the sieve with saline to remove any excess yeast using a spatula, gently pick up the embryos and place them in a small glass vial.Add about one milliliter of saline solution. Then cap the vial and incubate for five Minutes to wash the embryos. Following the Collection of embryos.Prepare the heptane fixed solution by combining 50%heptane and 50%fixative. The solution will be layered with heptane on top and the fixative on the bottom. After five minutes have passed, use a pasta pipette to remove the excess saline from the glass vial of embryos.Rinse the embryos with deionized water by applying approximately one milliliter of deionized water to the glass vial containing the embryos. Allow the embryos to sit for one minute before removing the water at a 50%bleach solution. Shake the vial a few times by hand and then incubate for three minutes in order to coate the embryos.The ated embryos will sink to the bottom of the vial. Embryonic CORs will float. Remove the bleach and the CORs after the three minute incubation.Following the decoration, rinse the embryos thoroughly with deionized water applying approximately one milliliter of deionized water to the glass vial containing the embryos. Allow the embryos to sit for one minute before removing the water leftover CORs will float and should be removed. Add the heptane fix solution to the embryos and incubate them for 30 minutes with gentle shaking to fix them following fixation.Use a pasta pipette to remove the fixative, which is the bottom layer, leaving the heptane in the tube. Then add methanol back to the bottom of the tube in place of the fixative and cap the tube shake vigorously by hand to remove the vitelline membrane. Following this step, the ized embryos will sink to the bottom of the tube.Using a pasta pipette, remove the methanol and hets from the vial leaving behind the deionized embryos. Then rinse the embryos five times with methanol, removing the methanol with a pasta pipette between each rinse. For each rinse, incubate the embryos for Five minutes.Place the embryos in a mixture of 50%PBT solution and 50%methanol for five minutes to rehydrate them. Then continue the rehydration process by transferring them to 100%PBT solution for five minutes. Next, block the embryos by incubating them in 5%BSA with 0.01%sodium azide solution for one hour at four degrees Celsius.Next, prepare the primary antibody for embryo staining by diluting it to the appropriate concentration using 5%BSA with 0.01%Sodium azide solution. The antibodies used here are directed against the vesicular cysteine string protein and anti HRP antibody. Anti HRP recognizes a neural specific carbohydrate moiety present in the surface glycoproteins, which labels all neurons.Incubate the embryos overnight at four degrees Celsius in the primary antibody solution. Wash the embryos 10 times with PBT over the course of an hour. By applying approximately one milliliter of PBT to the glass vial containing the embryos, allow the embryos to sit for six minutes before removing the PBT.Repeat this process of applying PBT, incubating and removing PBT 10 times. Prepare the secondary antibody solution in the dark because it is light sensitive by diluting to the appropriate concentration. We use Alexa secondary antibodies at a concentration of one to 200.Incubate the embryos for two hours at four degrees Celsius in the secondary antibody solution. Wrap the glass vial of embryos and secondary antibody solution in aluminum foil in order to avoid light exposure. Wash the embryos 10 times with PBT over the course of an hour.Add vector shield mounting medium to the embryos and incubate them overnight at four degrees Celsius to mount. Use a pastel pipette to suck up the embryos along with some of the mounting medium. Apply the embryos and medium to a glass slide.Apply a cover slip over the embryos and seal the cover slip with nail polish. Visualize embryos using a Fluorescence microscope here. Embryonic CNS is shown at stage 16 to 17.Cystine string protein is shown in red. HRP neurons are shown in green. Note the distinct staining of the commissure in this representative image.The embryonic PNS is shown at stage 16 to 17. Again, cystine string protein is shown in red, and neurons are shown in green. Note the repeated patterns of Peripheral neurons.When Attempting This procedure, it's important to be mindful of the embryos so as to not accidentally remove them from the vial. After watching this video, you should have a good understanding of how to prepare oph fla embryos for whole mount visualization of the nervous system.

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

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JoVE Journal

Neuroscience

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