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.
