Name: Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran (Video) | JoVE Journal
Uploaded: 2011-04-28T17:00:00.000+00:00
Duration: 7 min 35 s
Description: Learn lineage labeling in zebrafish embryos using UV-uncaging of caged fluorescein dextran and immunofluorescent microscopy. This method enables precise tracing of cell fates during development.

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5 minutes

We would like to thank Dan Carlin for help in making caged fluorescein. In addition we would like to thank the following funding sources: Vanderbilt University Medical School Program for Developmental Biology Training Grant to JAC, and NIH HD054534to JTG.

1. Synthesis of Caged Fluorescein Dextran
<ol>
	<li>Measure out 3.5 - 4 mg of aminodextran and add into the Invitrogen-supplied tinted tube containing 1 mg of CMNB-caged fluorescein SE. In our hands this ratio gives an average loading of ~ 2.5 dye molecules per dextran.</li>
	<li>Add 500 μL of 0.1 M Na2B4O7 (sodium borate) buffer to the tube.</li>
	<li>Cap and vortex for 30 seconds to dissolve the aminodextran and caged fluorescein.</li>
	<li>Let react overnight on a vortexing mixer.</li>
	<li>Twist off the bottom closure on a Zeba spin column and loosen the cap. Mark a dot on the column with a felt-tip pen. Place column in a 15-mL conical tube.</li>
	<li>Centrifuge at 1000 x g for 2 min with the dot facing the center of the rotor. Use column immediately after compacting.</li>
	<li>Place compacted column in a new 15-mL conical tube.</li>
	<li>Pool reaction mixture and transfer to the center of the compacted column resin bed. Replace cap.</li>
	<li>Centrifuge at 1000 x g for 2 min with the dot facing the center of the rotor. After the spin, the eluent and the top of the column will be roughly the same shade of yellow in color.</li>
	<li>Transfer the yellow eluent (~350 μL) to a 1.5-mL microcentrifuge tube.</li>
	<li>Lyophilize to dryness in a speedvac. Cover the speedvac with foil.</li>
	<li>Re-suspend the resulting ~ 2-3 mg of pale yellow foam in water to a final concentration of 1% w/v.</li>
	<li>Store this solution at -20 °C in foil-covered tubes. It is a good idea to aliquot the dextran solution to prevent repetitive freeze-thawing.</li>
</ol>
2. Injection of Caged Fluorescein Dextran Into Zebrafish Embryos
<ol>
	<li>Dilute 1% w/v stock of caged fluorescein dextran 1:5 or 1:10 in 0.2M KCl.</li>
	<li>Inject 0.5 - 1.0nL of caged fluorescein solution into the yolk of one cell stage embryos using a pressure injector. Once injected, clutches of embryos must be kept in the dark at 28.5°C to reduce non-specific uncaging.</li>
	<li>Manually remove chorions from embryos.</li>
	<li>Anesthetize embryos in 4% tricaine.</li>
	<li>Embryos will be mounted in 0.8% agarose in 35mmx10mm petri dish, then covered with egg water.</li>
	<li>Equilibrate melted agarose in 50°C heat block for at least 15 minutes.</li>
	<li>Using a glass pipette, draw up one embryo in a minimal amount of water and release it into the agarose. Note: It is important to cool the tube of agarose in your hand for approximately 30 seconds prior to putting the embryo into the tube to avoid killing the embryo.</li>
	<li>Pipette the embryo from the tube, along with approximately 50μL of agarose and eject the contents onto the center of the petri dish.</li>
	<li>Quickly orient the embryo using a plastic thread with the cells to be uncaged facing up. Note: The agarose with cool quickly so be hasty with orienting the embryo. Embryos can be damaged if manipulated after the agarose becomes rigid.</li>
	<li>Allow the agarose to completely solidify (this takes a few minutes) and fill the dish two thirds full with egg water containing 4% tricaine and 0.003% PTU.</li>
</ol>
3. Laser Uncaging of Fluorescein Dextran
<ol>
	<li>The microscope used for uncaging must have a 40X water immersion objective.</li>
	<li>We use a Photonics Instruments laser with the 365 nm dye cell and the 70%/30% split dichroic mirror.</li>
	<li>Prior to uncaging, the laser must be made parfocal with the objective and attenuated to the appropriate power.</li>
	<li>To focus the laser, place a mirrored glass slide under the objective and focus the objective until scratches in the slide are visible.</li>
	<li>Adjust laser attenuator until the laser can produce a clearly visible scratch in the mirror with a single pulse.</li>
	<li>Adjust the objective focus up and down. If the laser can produce a scratch when the objective is out of focus, adjust the laser focus ¼ turn up or down. Repeat until the laser can scratch the mirror only when the objective is focused.</li>
	<li>Place mounted embryo under the objective and focus on the area to be uncaged.</li>
	<li>To uncage, focus on a cell of interest and pulse it 10-20 times at 2-3 Hz.</li>
	<li>After uncaging, free the embryo by peeling agarose away with forceps and putting them in egg water containing 0.003% PTU. Let embryos develop in a light-tight box until they are fixed.</li>
</ol>
4. Antibody Labeling and Detection of Uncaged Fluorescein Dextran
<ol>
	<li>Fix embryos in Ab fixative (4% paraformaldehyde, 0.3mM CaCl2, 8% sucrose, 1X Phosphate Buffered Saline, pH 7.3) for two to four hours at room temperature (RT) or overnight (O/N) at 4°C. Keep embryos in a light tight box or foil-wrapped tube for all labeling steps.</li>
	<li>Remove fixative and replace with 100% MeOH, leave for 10 minutes at RT, remove MeOH and replace with fresh 100% MeOH.</li>
	<li>Store in MeOH at -20°C for at least 30 minutes. Note: You can store in MeOH for up to two weeks before epitopes begin to degrade.</li>
	<li>Rehydrate embryos with 5 minute washes at RT of 75% MeOH/1X Phosphate buffered saline with 0.1% Tween 20 (1X PBSTw), then 50% MeOH/50% 1XPBTw, then 25% MeOH/75% 1X PBSTw.</li>
	<li>Wash three times for 5 minutes in 1XPBSTw.</li>
	<li>If embryos are &gt;24 hours old, permiabilize embryos with 10 μg/mL proteinase K in 1XPBSTw. Permiabilization time is dependent on stage. Five minutes in proteinase K is sufficient if the embryos are between 24 and 48 hours post-fertilization (hpf). More time may be required if for older larvae. Re-fix in Ab fixative for 20 minutes at RT</li>
	<li>Wash five times for 5 minutes in 1XPBSTw at RT.</li>
	<li>Incubate in block solution (1XPBS, 0.1% Triton x100, 10% sheep serum, 10% Bovine serum albumen, 1% Dimethyl sulfoxide) for 1-2 hours at RT.</li>
	<li>Dilute primary antibodies (anti-fluorescein plus antibodies against cell-type specific markers) in blocking solution.</li>
	<li>Incubate embryos O/N at 4°C in solution containing primary antibodies.</li>
	<li>Wash four times for 20 minutes in 1XPhosphate Buffered Saline with 0.1% Triton x100 (PBSTx) at RT.</li>
	<li>Dilute fluorescently-labeled secondary antibodies (usually 1:300) in blocking solution.</li>
	<li>Incubate O/N at 4°C in solution containing secondary antibodies.</li>
	<li>Wash four times for 20 minutes in 1XPBSTx at RT.</li>
	<li>Clear embryos in 50% glycerol/50% 1XPBS for at least two hours at RT, then remove glycerol/PBS mixture and add 100% glycerol and let stand O/N at 4°C.</li>
</ol>
5. Representative Results:
After immunolabeling, there should be an enriched area of staining in the cells that were uncaged (Figure.1). It is not unusual to see a relatively high signal to noise ratio in &gt;48 hour old zebrafish, due to spontaneous uncaging of the fluorescein.
<img src="/files/ftp_upload/2672/2672fig1.jpg" alt="foxd3:GFP and fluorescein markers, dorsal view fluorescence microscopy; gene expression analysis." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. Lineage labeling of zebrafish pineal complex. At 24 hpf, a portion of the anterior pineal anlage, indicated by foxd3:GFP expression, was uncaged using UV laser pulses. A) By 48hpf, a left-sided cluster of cells called the parapineal (red circle) has emerged from the uncaged domain (white circle). B) Uncaged fluorescein signal is enriched in the parapineal (red circle) and the pineal organ (white circle).

<ol><li>Hatada, Y., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+fate+map+of+the+epiblast+of+the+early+chick+embryo%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">A fate map of the epiblast of the early chick embryo.</a> Development. 120, 2879-2889 (1994).</li>
<li>Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fates+of+the+blastomeres+of+the+32-cell-stage+Xenopus+embryo%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Fates of the blastomeres of the 32-cell-stage Xenopus embryo.</a> Dev Biol. 122, 300-319 (1987).</li>
<li>Wetts, R., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multipotent+Precursors+Can+Give+Rise+to+All+Major+Cell-Types+of+the+Frog+Retina%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Multipotent Precursors Can Give Rise to All Major Cell-Types of the Frog Retina.</a> Science. 239, 1142-1145 (1988).</li>
<li>Wetts, R., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microinjection+of+fluorescent+tracers+to+study+neural+cell+lineages%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Microinjection of fluorescent tracers to study neural cell lineages.</a> Development. , Suppl 2. 1-8 (1991).</li>
<li>Woo, K., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Order+and+coherence+in+the+fate+map+of+the+zebrafish+nervous+system%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Order and coherence in the fate map of the zebrafish nervous system.</a> Development. 121, 2595-2609 (1995).</li>
<li>Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+optical+marker+based+on+the+UV-induced+green-to-red+photoconversion+of+a+fluorescent+protein%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.</a> Proc Natl Acad Sci U S A. 99, 12651-12656 (2002).</li>
<li>Sato, T., Takahoko, M., Okamoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HuC%3AKaede%2C+a+useful+tool+to+label+neural+morphologies+in+networks+in+vivo%5BTitle%5D)+AND+%22Genesis%22%5BJournal%5D)">HuC:Kaede, a useful tool to label neural morphologies in networks in vivo.</a> Genesis. 44, 136-142 (2006).</li>
<li>Wiedenmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((EosFP%2C+a+fluorescent+marker+protein+with+UV-inducible+green-to-red+fluorescence+conversion%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion.</a> Proc Natl Acad Sci U S A. 101, 15905-15910 (2004).</li>
<li>Haugland, R. P. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHaugland%2C+RP+%22Handbook+of+Fluorescent+Probes+and+Research+Chemicals%22">Handbook of Fluorescent Probes and Research Chemicals</a>. , 9th Edition, 707-711 (2002).</li>
<li>Kozlowski, D. J., Murakami, T., Ho, R. K., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regional+cell+movement+and+tissue+patterning+in+the+zebrafish+embryo+revealed+by+fate+mapping+with+caged+fluorescein%5BTitle%5D)+AND+%22Biochem+Cell+Biol%22%5BJournal%5D)">Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein.</a> Biochem Cell Biol. 75, 551-562 (1997).</li>
<li>Kozlowski, D. J., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photoactivatable+%28caged%29+fluorescein+as+a+cell+tracer+for+fate+mapping+in+the+zebrafish+embryo%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Photoactivatable (caged) fluorescein as a cell tracer for fate mapping in the zebrafish embryo.</a> Methods Mol Biol. 135, 349-355 (2000).</li>
<li>Russek-Blum, N., Nabel-Rosen, H., Levkowitz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+resolution+fate+map+of+the+zebrafish+diencephalon%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">High resolution fate map of the zebrafish diencephalon.</a> Dev Dyn. 238, 1827-1835 (2009).</li>
<li>Summers, R. G., Piston, D. W., Harris, K. M., Morrill, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+orientation+of+first+cleavage+in+the+sea+urchin+embryo%2C+Lytechinus+variegatus%2C+does+not+specify+the+axes+of+bilateral+symmetry%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">The orientation of first cleavage in the sea urchin embryo, Lytechinus variegatus, does not specify the axes of bilateral symmetry.</a> Dev Biol. 175, 177-183 (1996).</li>
</ol>

No conflicts of interest declared.

As described, this protocol provides a relatively quick lineage labeling method in zebrafish that is built upon the commonly used techniques of microinjection, microscopy, and immunofluorescence. We have found that laser uncaging to be the most efficient and cost effective way to uncage fluorescein in a localized fashion. This method could be used to lineage label with experimental endpoints as late as 4 dpf. However, as cells divide, the caged-fluorescein concentration per cell eventually decreases beyond detectable lev...

A correction was made to <a href="http://www.jove.com/details.php?id=2672">Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran</a>. There was an error in the authors, Ilya A. Shestopalov and James K. Chen, names. The author's names have been corrected to: Ilya A. Shestopalov and James K. Chen instead of: Ilya Shestopalov and James Chen

A correction to the article Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran was made.

Erratum: Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

<table><tbody><tr><td>CMNB-caged fluorescein SE</td><td>Invitrogen</td><td>C20050</td><td></td></tr><tr><td>Aminodextran, 10kDa</td><td>Invitrogen</td><td>D1860</td><td></td></tr><tr><td>Zeba desalt spin columns</td><td>Pierce, Thermo Scientific</td><td>89889</td><td></td></tr><tr><td>goat anti-fluorescein antibody</td><td>Invitrogen</td><td>A11095</td><td></td></tr><tr><td>Alexa Fluor 633 Donkey anti-goat antibody</td><td>Invitrogen</td><td>A21082</td><td></td></tr><tr><td>35mmx10mm petri dishes</td><td>BD Biosciences</td><td>351008</td><td></td></tr><tr><td>Upright compound microscope with 40x water immersion objective</td><td>Leica Microsystems</td><td>DM6000B</td><td></td></tr><tr><td>MicroPoint Manual Laser Illumination System</td><td>Photonic Instruments</td><td></td><td></td></tr><tr><td>Phenylthiourea (PTU)</td><td>Alfa Aesar</td><td>L06690</td><td></td></tr><tr><td>Tricaine</td><td>Sigma-Aldrich</td><td>E10521</td><td></td></tr><tr><td>Proteinase K</td><td>Roche Group</td><td>03115836991</td><td></td></tr><tr><td>Plastic thread</td><td>Any Supplier</td><td></td><td></td></tr><tr><td>Agarose</td><td>Research Products International Corp.</td><td>A20090</td><td></td></tr><tr><td>SpeedVac</td><td>Thermo Fisher Scientific, Inc.</td><td>Savant SPD131DDA</td><td></td></tr><tr><td>Vortexing mixer</td><td>VWR international</td><td>Vortex Genie 2</td><td></td></tr><tr><td>Pressure injector</td><td>Applied Scientific Instrumentation</td><td>MPPI-3</td><td></td></tr></tbody></table>

lineage labeling of zebrafish cells with laser uncagable fluorescein dextran

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

The overall goal of this procedure is to lineage label cells in a zebrafish embryo. This is accomplished by first synthesizing caged fluorescein and injecting it into the zebrafish embryos. Embryos are then mounted in agarro so that the cells to be labeled are accessible.Next, a UV laser is used to activate the caged fluorescein in selected cells of the embryo. Finally, embryos are harvested after they have developed to the desired stage and the labeled cells are detected in relation to markers of particular lineages. Ultimately, results can be obtained that show the fate of selected cells in the embryos through photo uncaging and immunofluorescent microscopy.The main advantage of this technique over existing techniques such as photo conversion of Cade, is that multicolored immunofluorescence can detect uncaged fluorescein in addition to other cell tie specific antibodies. To inject caged fluorescein dextran into zebrafish embryos, first synthesize it and prepare 1%weight per volume. Stock aliquots according to the accompanying written protocol.Next, dilutant aliquot, one to five or one to 10 in 0.2 molar potassium chloride. Next, inject 0.5 to one nanoliter of caged fluorescein solution into the yolk of one cell stage embryos. Using a pressure injector once injected, clutches of embryos must be kept in the dark at 28.5 degrees Celsius to reduce non-specific uncaging at the desired developmental stage.Manually remove the choon from the embryos, anesthetize the embryos in 4%trica to mount the embryos equilibrate, 0.8%melted aros in a 50 degree Celsius heat block for at least 15 minutes. Then cool the tube in your hand for approximately 30 seconds Prior to adding embryos using a glass pipette, draw up one embryo in a minimal amount of water and release it into the aros. Pipette the embryo from the tube along with approximately 50 microliters of aros and expel the contents onto the center of a 35 millimeter by 10 millimeter Petri dish.Using a plastic thread, quickly orient the embryo with the cells to be uncaged facing up. Allow the aros to completely solidify and fill the dish. Two thirds full with egg water containing 4%trica and 0.003%PTU keep embryos in a light tight box until on caging.To perform laser on caging of fluorescein dextran, use a compound microscope with a 40 x water immersion objective lens. We use a photonics instruments laser with a 365 nanometer dye cell and a 70%30%split dichroic mirror prior to uncaging. The laser must be made par focal with the objective and attenuated to the appropriate power to focus the laser.Place a mirrored glass slide under the objective and focus the objective until scratches in the slide are visible. Adjust the laser attenuator until the laser produces a clearly visible scratch in the mirror with a single pulse, adjust the objective focus up and down. If the laser can produce a scratch when the objective is out of focus, adjust the laser focus one quarter.Turn up or down. Repeat until the laser can scratch the mirror. Only when the objective is focused, place mounted embryo under the objective and focus on the area to be uncaged to uncage.Focus on a cell of interest and pulse it 10 to 20 times at two to three hertz. After uncaging. Free the embryo by peeling the aeros away with forceps and putting them in egg water containing 0.003%PTU Fix embryos in antibody fixative for two to four hours at room temperature or overnight at four degrees Celsius.Remove the fixative and replace it with 100%methanol. Incubate for 10 minutes at room temperature. Then remove the methanol and replace it with fresh 100%methanol.Store the embryos in methanol at minus 20 degrees Celsius for at least 30 minutes. You can store in methanol for up to two weeks before epitopes begin to degrade. Rehydrate the embryos with five minute washes at room temperature of 75%Methanol 25%one x phosphate buffered saline with 0.1%tween 20 50%methanol 50%one XPBS tween 20, then 25%methanol, 75%one XPBS tween 20 wash three times for five minutes in one XPBS tween 20.If embryos are greater than 24 hours old, perme them with 10 micrograms per milliliter. Proteinase K in one XPBS tween 20 permeation time is dependent on the embryo stage. Five minutes in protease K is sufficient.If the embryos are between 24 and 48 hours post fertilization, more time may be required for older larvae. Refix an antibody fixative for 20 minutes at room temperature. Wash the embryos five times for five minutes in one XPBS tween 20 at room temperature.Then incubate in block solution for one to two hours at room temperature. Dilute primary antibodies in blocking solution. Then incubate embryos overnight at four degrees Celsius in solution containing primary antibodies the next morning while washing the embryos four times in one XPBS tritton X 100 dilute fluorescently labeled secondary antibodies in blocking solution.Incubate the embryos overnight at four degrees Celsius in solution containing secondary antibodies. After washing four times in one XPBS Triton X 100, clear the embryos in 50%glycerol 50%one XPBS for at least two hours at room temperature. Then remove the glycerol PBS mixture and add 100%glycerol incubate overnight at four degrees Celsius.Represen results for control and photo uncaged larvae are shown in the form of confocal projections. Control embryos were injected with caged fluorescein but not subjected to photo uncaging at 48 hours post fertilization. The control embryo exhibits GFP labeling in the pineal complex.Some punctate background labeling, but no localized accumulation of labeled fluorescein is detectable. Photo uncaged embryos show clear accumulation of uncaged fluorescein in some of the GFP labeled cells. These are the descendants of the cells targeted by photo on caging 24 hours earlier With minor modifications.This procedure can be used to lineage label almost any tissue in the zebrafish embryo prior to 48 hours post fertilization.

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Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

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