Name: Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo (Video) | JoVE Journal
Uploaded: 2009-09-15T13:00:00.000+00:00
Duration: 9 min 25 s
Description: Learn microinjection techniques for studying mitosis in Drosophila melanogaster syncytial embryos. This method enables precise manipulation and high-resolution imaging of cellular division processes.

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

This protocol is currently used in our laboratory and has been refined over the years by many people including Drs David Sharp, Mijung Kwon, Patrizia Sommi, Dhanya Cheerambathur. We thank Dr Bill Sullivan (UCSC) who provided us with excellent advice on the manipulation and microinjection of early Drosophila embryos when our work in this system was being initiated (ref. 13). We thank all members of the Scholey laboratory. Our work on mitosis in Drosophila is supported by NIH grant GM55507.

Recipes:
Grape Juice plates:
<ul>
<li>5.5g bacto agar</li>
<li>14.5 g dextrose or glucose</li>
<li>7.15 g sucrose</li>
<li>45 ml grape juice concentrate (100% juice).</li>
<li>204.5 ml H20</li>
<li>625 μl 10N NaOH</li>
</ul>
Mix all ingredients and microwave until boiling. Add 2.8 ml acid mix (acid mix: 20.9 ml propionic acid, 2.1 ml phosphoric acid, 27 ml H20)
Mix and pour onto 35 mm Petri dishes. Let solidify at room temperature for one or two days. If plates are not going to be used soon, seal with parafilm and keep at 4°C (allow to equilibrate to RT before using).
Yeast paste:
Dissolve about one teaspoon of yeast (Sigma YSC2, yeast from Saccharomyces cerevisiae type II) in water to form a thick paste. Place a small amount of this on each grape juice plate just prior to use.
G-PEM buffer:
<ul>
<li>80 mM Na-PIPES, pH 6.9</li>
<li>1 mM MgCl2</li>
<li>1 mM EGTA</li>
<li>1 mM GTP</li>
</ul>
Injection buffer:
<ul>
<li>150 mM K-Aspartate</li>
<li>10 mM K-phosphate</li>
<li>20 mM imidazole, pH 7.2</li>
</ul>
Heptane glue:
Unroll double sticky tape and place in a 100ml bottle, add about 50ml heptane, seal the bottle, and rock for several days. When using, add heptane if the glue is too thick.
Dehydration chamber:
Take a 100 mm Petri dish, put one part of a 35 mm dish inside to make a "table" and add Drierite (anhydrous calcium sulfate) around it so that the height of Drierite is no higher than the "table" and cover. The coverslip with embryos will be placed on this "table" for dehydration before injection. It is a good idea to use at least some indicating Drierite and change it when it has changed color.
Protocol:
This protocol can be used for injection of virtually any soluble reagent into the Drosophila syncytial embryo: For example, either a fluorescent protein for observation or a target protein inhibitor or both.
Getting everything ready:
1.- 2 to 3 days before experiment, make grape juice plates and set up lay cage: Take a plastic bottle (6 oz, round bottom from Applied Scientific Drosophila), cut two small holes (about 1cm2) on opposite sides of the bottle and fill with a cotton piece (this will allow air to flow), tap the flies into the bottle and cover with a grape juice plate. Keep at 25°C (or room temperature), flies should lay embryos for about 10 days.
2.- At least one day before the experiment pull needles for injection, we use a Kopf Needle/Pipette puller model 720 and thin walled glass filaments (World Precision Instruments tw100f). For injection of fluorescent tubulin, keep needles at 4°C to prevent temperature-induced polymerization in the needle.
Tubulin preparation:
Note: All work with tubulin should be done at 4°C to prevent polymerization.
<ol>
<li>Take labeled tubulin out of the freezer and put it on ice for 5 to 15 minutes.</li>
<li>Dilute with G-PEM buffer (4 to 10 fold depending on the intensity required).</li>
<li>Centrifuge at 13k rpm at 4°C for 10-15 minutes.</li>
<li>Load 1 - 2 μl into needle.</li>
</ol>
Antibody or chemical inhibitor:
Prepare needles as for tubulin, using an appropriate concentration (this may need to be tested). The antibody can be in injection buffer, G-PEM buffer or PBS. If another buffer is needed, this buffer will need to be injected as a control to make sure this buffer causes no damage on its own.
Note: Inhibitor and fluorescent tubulin can be mixed and injected together if the concentrations allow it. Otherwise, inject the fluorescent tubulin and wait 5 to 10 minutes before injecting the inhibitor. When performing a double injection, start with more embryos, since fewer embryos will recover normally.
Embryo collection:
<ol>
<li>Put a new grape juice plate on the lay cage.</li>
<li>Remove this plate after one hour, this is the first collection of the day and often is not very good. It can be discarded.</li>
<li>Change the plate every hour to keep collecting embryos for one hour each.</li>
</ol>
Coverslip preparation:
<ol>
<li>Put a 50 x 22mm coverslip on one side of a microscope slide. Tape the four corners to the slide so it doesn't move.</li>
<li>Using a cotton tipped applicator, put one layer of heptane glue in one line on the coverslip (the glue should not be viscous and should dry in a few seconds, otherwise add more heptane).</li>
<li>Put a piece of double sticky tape on the slide toward the side of the coverslip.</li>
</ol>
Embryo preparation:
Note: Embryos should be imaged about 2 hours after start of collection, so begin the following steps allowing sufficient time to finish them within this time-frame.
<ol>
<li>With a moistened brush carefully pick up the embryos from the grape juice plate and place on double sticky tape on slide.</li>
<li>Using the outer part of the tweezers, roll the embryos over the double sticky tape until the chorion (the outer membrane) breaks open.</li>
<li>Pick up the embryo by gently rolling it over the chorion so it sticks to the tweezer and place it on the heptane glue on the coverslip with the long side of the embryo parallel to the long side of the coverslip. Set up 10 to 20 embryos in one row.</li>
<li>Remove the coverslip and place it in the dehydration chamber for 3 to 8 minutes (this depends on the humidity of the room and the amount to be injected).</li>
<li>Place on a metal chamber with vacuum grease.</li>
<li>Cover the embryos with halocarbon oil 700 to avoid further dehydration. Embryos are now ready for injection.</li>
</ol>
Embryo injection:
<ol>
<li>Find the embryos under a 16x objective.</li>
<li>Move the embryos away, and find the needle without moving the focal plane.</li>
<li>Center the needle and move it up without moving it in x or y direction.</li>
<li>If the needle is not open, put the edge of the coverslip in the field of view, but not where the needle will be, and lower the needle to the same focal plane. Very carefully move the coverslip until it hits the needle and gently breaks it open. If the needle is open, go to step 5. (Needles can be opened with hydrofluoric acid before filling).</li>
<li>Put the embryos in view (but not where the needle will be), lower the needle into the oil and make sure you obtain nice liquid drops from the needle.</li>
<li>Carefully but steadily move the embryo into the needle and inject a drop into the embryo and move the embryo away. (Or follow the instructions of your injection apparatus).</li>
<li>After all the embryos have been injected, they are ready for observation on a confocal microscope.</li>
</ol>
Imaging:
Image the embryos on a confocal microscope with a 60 or 100X objective: Notes:
<ol>
<li>The time interval on a time lapse series should be at most a few seconds, since mitosis in the embryo is very fast, depending on the exact process to be studied.</li>
<li>A 3D series or a single plane can be acquired depending on the process of interest.</li>
<li>For fast-acting inhibitors, the transfer from injection microscope to imaging microscope has to be fast.</li>
</ol>
<img src="/files/ftp_upload/1382/1382fig1.jpg" alt="Cell division process; spindle diagram and microscopy; spindle length vs. time graph; microscopic images." data-altupdatedat="1787630408000" data-alt="figure 1"> Figure 1: Study of mitosis in the Drosophila syncytial embryo. Nuclei in the early embryo undergo rapid divisions without cytokinesis, during cycles 10 thru 13 nuclei form a monolayer at the cortex. A. Schematic of early embryo with nuclei at cortex. Embryo can express GFP and/or RFP labeled proteins and can be microinjected with other labeled proteins and/or inhibitors. B. Time lapse imaging of embryo expressing GFP-tubulin and RFP-histone. C. Plot of pole-pole separation for cycles 11, 12 and 13 in a wild type embryo. Spindle length exhibits periods of isometric length and periods of elongation, which are very consistent and reproducible. D. Dynamics of spindle microtubules. Injection of low concentration of rhodamine tubulin leads to speckles formed of a few tubulin subunits, which can be used to study microtubule dynamics.

<ol><li>Brust-Mascher, I., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitotic+spindle+dynamics+in+Drosophila%5BTitle%5D)+AND+%22Int+Rev+Cytol%22%5BJournal%5D)">Mitotic spindle dynamics in Drosophila.</a> Int Rev Cytol. 259, 139-172 (2007).</li>
<li>Duffy, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GAL4+system+in+Drosophila%3A+a+fly+geneticist%27s+Swiss+army+knife%5BTitle%5D)+AND+%22Genesis%22%5BJournal%5D)">GAL4 system in Drosophila: a fly geneticist's Swiss army knife.</a> Genesis. 34, 1-15 (2002).</li>
<li>Morris, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microinjection+methods+for+analyzing+the+functions+of+kinesins+in+early+embryos%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Microinjection methods for analyzing the functions of kinesins in early embryos.</a> Methods Mol Biol. 164, 163-172 (2001).</li>
<li>Brust-Mascher, I., Sommi, P., Cheerambathur, D. K., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinesin-5-dependent+poleward+flux+and+spindle+length+control+in+Drosophila+embryo+mitosis%5BTitle%5D)+AND+%22Mol+Biol+Cell%22%5BJournal%5D)">Kinesin-5-dependent poleward flux and spindle length control in Drosophila embryo mitosis.</a> Mol Biol Cell. 20, 1749-1762 (2009).</li>
<li>Brust-Mascher, I., Civelekoglu-Scholey, G., Kwon, M., Mogilner, A., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Model+for+anaphase+B%3A+role+of+three+mitotic+motors+in+a+switch+from+poleward+flux+to+spindle+elongation%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Model for anaphase B: role of three mitotic motors in a switch from poleward flux to spindle elongation.</a> Proc Natl Acad Sci U S A. 101, 15938-15943 (2004).</li>
<li>Brust-Mascher, I., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microtubule+flux+and+sliding+in+mitotic+spindles+of+Drosophila+embryos%5BTitle%5D)+AND+%22Mol+Biol+Cell%22%5BJournal%5D)">Microtubule flux and sliding in mitotic spindles of Drosophila embryos.</a> Mol Biol Cell. 13, 3967-3975 (2002).</li>
<li>Cheerambathur, D. K., Brust-Mascher, I., Civelekoglu-Scholey, G., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+partitioning+of+mitotic+kinesin-5+crosslinkers+between+microtubule-bound+and+freely+diffusing+states%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Dynamic partitioning of mitotic kinesin-5 crosslinkers between microtubule-bound and freely diffusing states.</a> J Cell Biol. 182, 421-428 (2008).</li>
<li>Cheerambathur, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+analysis+of+an+anaphase+B+switch%3A+predicted+role+for+a+microtubule+catastrophe+gradient%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Quantitative analysis of an anaphase B switch: predicted role for a microtubule catastrophe gradient.</a> J Cell Biol. 177, 995-1004 (2007).</li>
<li>Kwon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+chromokinesin%2C+KLP3A%2C+dives+mitotic+spindle+pole+separation+during+prometaphase+and+anaphase+and+facilitates+chromatid+motility%5BTitle%5D)+AND+%22Mol+Biol+Cell%22%5BJournal%5D)">The chromokinesin, KLP3A, dives mitotic spindle pole separation during prometaphase and anaphase and facilitates chromatid motility.</a> Mol Biol Cell. 15, 219-233 (2004).</li>
<li>Sharp, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+coordination+of+three+mitotic+motors+in+Drosophila+embryos%5BTitle%5D)+AND+%22Mol+Biol+Cell%22%5BJournal%5D)">Functional coordination of three mitotic motors in Drosophila embryos.</a> Mol Biol Cell. 11, 241-253 (2000).</li>
<li>Sharp, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+bipolar+kinesin%2C+KLP61F%2C+cross-links+microtubules+within+interpolar+microtubule+bundles+of+Drosophila+embryonic+mitotic+spindles%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">The bipolar kinesin, KLP61F, cross-links microtubules within interpolar microtubule bundles of Drosophila embryonic mitotic spindles.</a> J Cell Biol. 144, 125-138 (1999).</li>
<li>Sharp, D. J., Rogers, G. C., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytoplasmic+dynein+is+required+for+poleward+chromosome+movement+during+mitosis+in+Drosophila+embryos%5BTitle%5D)+AND+%22Nat+Cell+Biol%22%5BJournal%5D)">Cytoplasmic dynein is required for poleward chromosome movement during mitosis in Drosophila embryos.</a> Nat Cell Biol. 2, 922-930 (2000).</li>
<li>Sharp, D. J., Yu, K. R., Sisson, J. C., Sullivan, W., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antagonistic+microtubule-sliding+motors+position+mitotic+centrosomes+in+Drosophila+early+embryos%5BTitle%5D)+AND+%22Nat+Cell+Biol%22%5BJournal%5D)">Antagonistic microtubule-sliding motors position mitotic centrosomes in Drosophila early embryos.</a> Nat Cell Biol. 1, 51-54 (1999).</li>
<li>Waterman-Storer, C. M., Desai, A., Bulinski, J. C., Salmon, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescent+speckle+microscopy%2C+a+method+to+visualize+the+dynamics+of+protein+assemblies+in+living+cells%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells.</a> Curr Biol. 8, 1227-1230 (1998).</li>
</ol>

This protocol is relatively straightforward, however, each step requires practice to make sure the embryos are not damaged. Careful control experiments always need to be done to ensure that reliable results are being obtained. One good way to begin this is by microinjecting buffer or rhodamine tubulin into control embryos expressing GFP-tubulin to make sure that mitosis proceeds normally through all cycles at the embryo surface (cycles 10 through 13). In control embryos you should not observe any physical connections bet...

microinjection techniques for studying mitosis in the drosophila melanogaster syncytial embryo

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

This procedure begins with the collection of embryos on fresh grape juice plates 30 to 60 minutes. After removing the plate from the lake cage, pick up the embryos with a moistened brush and place them on double sticky tape. Gently roll one embryo over the tape using one half of a tweezer until the corion breaks open.Pick up the embryo and place it gently on the cover. Slip over a layer of het glue. Repeat this until you have enough embryos.After dehydration, the embryos are ready for injection. Hi, I'm Ingrid Bruce Musher from the laboratory of Dr.Jonathan Sculley in the Department of Molecular and Cellular Biology at the University of California Davis. Today we'll show you a procedure to micro inject drosophilas syncytial embryos.We use this procedure to study the role of micro ttes and mitral based motors in mitosis. For example, we micro inject antibodies or other inhibitors of specific mitotic proteins. By analyzing the inhibitor effect on mitosis, we are able to determine the target proteins function.So let's get started. To begin preparations two to three days before the experiment, set up the lay cage by cutting two small holes about one centimeter squared on opposite sides of a plastic bottle. Fill the holes with a cotton piece, allowing air to flow.Tap the flies into the bottle and then cover it with a grape juice plate. With yeast, keep the leg cage at room temperature flies will lay eggs for up to 10 days at least one day before the experiment. Pull needles for injection and keep them at four degrees Celsius.On the day of the experiment, prepare and load the needles. First, take the labeled tubulin out of the freezer and put it on ice for five to 15 minutes. Next, dilute it with GPEM buffer and then centrifuge it for 10 to 15 minutes at 13, 000 RPM four degrees Celsius.Finally, load one to two microliters into the needle and keep it at four degrees Celsius. To prevent temperature induced polymerization in the needle. Before setting up the cover slip, make heptane glue by enrolling double sticky tape and placing it in a 100 milliliter bottle.Add about 50 milliliters of heptane, seal the bottle and rock it for several days. Before preparing the embryos, make a dehydration chamber that the embryos will be placed in prior to injection. To do this, put one part of a 35 millimeter dish inside of a 100 millimeter Petri dish to make a table add dryer, right, which is anhydrous calcium sulfate around it so that the height of the dryer right is no higher than the table and cover.To begin preparing the embryos, first, collect them from the lake cage by changing the grape juice plate with yeast every hour, the embryos should be imaged about two hours after the start of the collection. To prepare the cover slip, place it on one side of a microscope, slide and tape the four corners down so it doesn't move. Using a cotton tipped applicator, put one layer of heptane glue in a line on the cover slip.The glue should not be viscous and should dry in a few seconds. If it is too thick, add more heptane. Finally, put a piece of double sticky tape on the slide next to the cover.Slip with a moistened brush. Carefully pick up the embryos from the grape juice plate and place them on the double sticky tape on the slide. Next, use one half of a tweezer to roll the embryos over the double sticky tape until the corion breaks open.Pick up the embryo by gently rolling it over the Corian so it sticks to the tweezer and place it on the heptane glue on the cover. Slip with the long side of the embryo parallel to the long side of the cover slip. Place 10 to 20 embryos in one row.Remove the cover slip and place it in the dehydration chamber for three to eight minutes. The time depends on the local humidity and the amount to be injected. Next, place the cover slip on a metal chamber with vacuum grease.Finally, cover the embryos with halo carbon oil to avoid further dehydration. The embryos are now ready for injection. First, find the embryos under a 16 x objective.Move the embryos away and find the needle without moving the focal plane. Center the needle and move it up without moving it in the X or Y direction. To open the needle, put the edge of the cover slip into the field of view, but not where the needle will be.And lower the needle to the same focal plane. Very carefully move the cover slip until it hits the needle and gently breaks it open. Move the needle up.Now bring the embryos into view. Lower the needle into the oil, and to make sure to obtain nice liquid drops from the needle. Steadily move the embryo into the needle, inject a drop into the embryo, and then move the embryo away.After all the embryos have been injected, they are ready for observation on a confocal microscope with a 60 or 100 x objective. A control embryo should look like the one in this movie. This is an embryo expressing GFP tubulin and RFP histone imaged with a 100 x objective on a spinning disc confocal microscope.The total time shown is eight minutes. Plots of pole to pole distance are very reproducible and are a reliable measure of success in control embryos. They should look like those shown here for cycles 11 through 13.Shown in this movie is another control embryo. This one expressing KLP 61 FGFP and injected with r Domine tubulin. This was also imaged with a 100 x objective on a spinning disc confocal, and the total time is seven minutes.This movie was acquired immediately after Microinjection, and the gradient in the concentration of r Domine tubulin is clearly visible at the start of the movie. These embryos can be used to study the inhibition of Klp 61 F after microinjection of Klp 61 F antibody. We can see the loss of Klp 61 FGFP from the spindles.We've just shown you how to micro inject drosophilas incisional embryos. When doing this procedure, it is important to remember to perform controls, first practice injecting buffer or rutin alone, and make sure mitosis proceeds normally through cellular. Once all your injections are reliable and not causing damage, you can inject an inhibitor and study the effect of that particular inhibition.So that's it. Thanks for watching and good luck with your experiments.

microinjection-techniques-for-studying-mitosis-drosophila

Research

JoVE Journal

Biology

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

Due to their large size and relatively simple organization, Drosophila nurse cells are ideally suited for live cell imaging studies. This video describes a microinjection method for exogenous compounds delivery into the cells, and the featured protocol demonstrates the procedure with fluorescent oligonucleotide probes called molecular beacons (MBs) used to visualize endogenous mRNA transcripts.

Microinjection of Drosophila Nurse Cells: A Method of Intracellular Delivery

This video describes how to perform microinjections that deliver reagents into live Drosophila embryos. Researchers can use this method to introduce exogenous compounds like nucleic acids and proteins during the early stages of embryo development. The featured protocol demonstrates how to set up and perform the procedure for injection of any soluble reagent–here, a fluorescent protein for live imaging experiments.

Microinjection of Live Drosophila Embryos: Early Delivery of Reagents to the Developing Embryo

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

The goal of this protocol is to inject Rhodamine-conjugated globular actin into Drosophila embryos and image intranuclear actin rod assembly following heat stress.

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

Presented here is a protocol to microinject and simultaneously image multiple Drosophila embryos during embryonic development using a plate-based, high content imager.

Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer

This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents an optimized protocol for embryo microinjection, a crucial step for creating transgenic flies.

Embryo Microinjection for Transgenesis in Drosophila

Here, we provide a detailed protocol to perform live imaging of the asymmetric division of germline stem cells (GSCs) in the Drosophila ovarian niche. We use a transgenic line that ubiquitously expresses a green fluorescent protein (GFP) fusion of the spectrosome protein Par-1.

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

These protocols outline how to characterize the organization and occluding function of pleated septate junctions in control and mutant Drosophila embryonic epithelia.

Immunostaining and Dye Penetration Experiments to Define Core Pleated Septate Junction Proteins in Drosophila Embryonic Epithelia

Learn microinjection techniques for studying mitosis in Drosophila melanogaster syncytial embryos. This method enables precise manipulation and high-resolution imaging of cellular division processes.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

Explore More Videos