The overall goal of the following experiment is to image neural cells at high resolution In the zebra fish embryo first, DNA encoding membrane targeted green fluorescent protein is injected into a one cell stage zebra fish embryo, which results in mosaic inheritance, thereby enabling enhanced imaging of scattered cells. Embryos are then immuno labeled with anti GFP and sectioned enabling visualization of the morphology of single cells at high resolution in a fixed preparation. Alternatively, live embryos can be examined using time-lapse video microscopy in order to image cellular dynamics.
Results are obtained that show cell morphology or dynamics. Here, the zebrafish hind brain is imaged with confocal microscopy. Hello everyone.
I'm PR Chandran from the laboratory of Dr.Rachel Brewster in the biological sciences department at the University of Maryland Baltimore County. Today we'll show you a procedure to label and image neural cells in zebrafish embryo at higher resolution. We typically use this procedure in a laboratory to study the mechanisms of neuro relation in zebra fish embryo at a cellular level.
So let's get started. Place wet. Kim wipes around the edge of a 100 millimeter Petri dish.
Carefully secure a glass microinjection needle on a strip of wax inside the humidified dish. Under a dissecting stereo microscope, align the needle against a graduated slide. To obtain a tip of the appropriate size, use a clean razor blade to gently tap the needle where it has a one micrometer diameter.
Connect the needle to the microinjection apparatus pipette 0.5 microliters of a solution of phenol red and water onto para film and front fill the needle from the droplets. Set the microinjection pressure to seven kilo pascals and begin with a 20 millisecond pulse Count the number of pulses it takes to dispense all of the liquid. Vary the time between 10 and 30 milliseconds so that it takes approximately 250 pulses to dispense.
0.5 microliters of solution. Use a micro pipette to backfill the needle with 0.5 to one microliter of MG FPD NA in water, mixed with phenol red For visualization. Cover the humidified dish to prevent evaporation of the solution out of the needle under the stereo microscope.
Position one cell stage separate fish embryos in a 60 millimeter Petri dish filled with embryo.Medium. Use fine forceps to gently grip the Corian and stabilize the embryo. Move the needle filled with MGFP and phenol red into the targets area for injection.
For broad range MGFP expression inject into the yolk or the cytoplasm. Use the microinjection apparatus to deliver approximately two nanoliters of the MGFP solution. Observe the location of the phenol red indicator to confirm proper injection.
Carefully remove the needle from the embryo. Inject all the embryos in the dish up to 30 embryos at one time. Avoid exposing the embryos to light incubate at 28.5 degrees Celsius to allow the embryos to develop to the desired stage under the stereo microscope.
Use fine forceps to gently peel the Corian off the embryos. Use a pasta pipette to remove the empty corian from the dish, leaving only the dec coated embryos. Use a fire polished pasta pipette to transfer the injected coated embryos into a 1.5 milliliter micro centrifuge tube.
Remove as much extra medium as possible, then add one milliliter of ative. Wash the embryos in room temperature one XPB S3 times for five minutes each. Move the embryos to a petri dish filled with one XPBS under the stereo microscope.
Use fine forceps to hold the embryo near the tail region. Use a second set of forceps or a poker to try and remove as much yo as possible without touching the hind frame region. Looking under the stereo microscope, use the fine forceps to lift the embryo from the dish and into a sectioning mold filled with 4%low melting point.Aros.
Position the embryo towards one end of the mold. Allow the aros to solidify at room temperature. Use a spatula to remove the hardened aros block from the mold with a raise of blade cut off the bottom end of the block evenly.
Use a drop of super glue to anchor the block to the vibrato stage cut side down if desired. Align multiple blocks on the stage. Attach the stage to the vibrato.
Make sure the blade will slice all the way through each embryo, but not completely through the block. Fill the stage with one XPBS section. The embryos carefully cut to the back of one arou block and transfer it to a Petri dish filled with one XPBS.
Separate the sections under the stereo microscope. Use fine forceps to place the desired sections into a multi-well dish filled with one XPBS. Use a different well for each embryo.
Stain the sections with DPI and immuno label with anti GFP. If not, imaging within 48 hours. Mount up to six sections per slide and place a cover slip on top of the sections for confocal imaging.
Store the slides in the dark to avoid photo bleaching. The live imaging, the MGFP injected embryos must be protected from light. Use forceps to coate embryos 30 minutes before they reach the desired developmental stage.
Transfer the coated embryos to a sedative solution of embryo medium with 0.01%Trica incubate a room temperature for 15 minutes. Pipette drop of 1%regular arose in water on a 35 millimeter glass bottom culture dish so that it fills the mold. Allow the agros to solidify.
Use the 90 millimeter glass capillary tube to make three holes in the drop of aros under the stereo microscope. Examine the bottom of the well and remove any residual aros with forceps. Fill the dish with embryo medium under the stereo microscope.
Use a pasta pipette to transfer one embryo to each hole in the aros. Use fine forceps to orient the embryo so that the area of interest in this case the dorsal head is resting against the glass. Cover the dish.
Carefully transport the dish to the stage of a confocal microscope. Use space heaters to keep the embryos at 28.5 degrees Celsius throughout the imaging. Here is a section through the hind brain of a wild type embryo at four to five somites double labeled with membrane targeted GFP in green and D in blue only one cell is labeled with membrane bound GFP allowing high resolution imaging of cellular morphology and organization.
This is a comparable image of the hind brain of an nardi din mutant at for to five somites. This image reveals that the cells in nardi and mutants fail to orient properly towards the midline during neuro revelation. In contrast to the method for marking cells with MGFP labeling of the hindbrain with a general cell surface marker such as beta-catenin does not allow the morphology of individual cells to be visualized.
Time-lapse imaging of the hind brain of an MG FP labeled embryo reveals that the cell behaviors that drive neuro relation in the zebra fish are very dynamic and involve the extension of membrane protrusions as cells migrate and inter collate. We have just shown you how to prepare both fixed and live zebra fish embryos for single cell imaging at high resolution. So that's it.
Thanks for watching and good luck with your experiments.
