The overall goal of this procedure is to induce precisely controlled damage to zebrafish, embryonic skeletal muscle in order to study muscle regeneration processes. This is accomplished by first genetically labeling single skeletal muscle cells in a mosaic fashion. Next selected embryos are embedded in agros supplied with trica on a slide.
Then the embryos are placed under the microscope and targeted muscle cells are injured using a micro point laser. Finally, the embryos are removed from the agros and allowed to recover for defined periods of time. Ultimately, results can be obtained that show changes in myo fibrillar organization through laser injury followed by confocal microscopy.
The method that we present today is relevant to the field of muscle regeneration, but can also be applied to other paradigms of regeneration such as fin regeneration and zebra fish To label cells inject one cell stage embryos with a plasmid encoding GFP or any GFP fusion protein under the control of ubiquitous or cell types specific promoter. During development, GFP is then expressed in a mosaic fashion within tissues. In this example, the plasmid injected causes single muscle cells to express alpha actin in GFP.
Under the control of the beta actin promoter incubate the embryos until 28 hours post fertilization. Next under a dissection microscope fitted with a fluorescent lamp for detecting actinin. GFP clones use forceps to coate the embryos make a 1%low melting point, agros 10%trica solution with E three medium by boiling 100 milligrams of agros in 10 milliliters of E three medium.
Let the solution cool to 39 degrees Celsius and add trica to a final concentration of 10%Then prepare a microscope slide with a petroleum jelly ring. After adding low melting point agro solution to the petroleum jelly ring under the microscope, embed a single embryo in the low melting point agros within the petroleum jelly ring. With a cover slip, gently cover the embryo and parallel to the cover slip for a perfect lateral view.
To prepare the micro point laser, please refer to the micro point laser manual for detailed instructions on how to set up and use the laser. In conjunction with the microscope, select the CUMERIN four 40 laser dye for the generation of a 435 nanometer wavelength laser beam. Using the attenuation slider, adjust the laser power.
The laser power required for celebration should be strong enough to break a glass surface with a single pulse. Refer to the manufacturer's instructions for additional details on adjusting the laser using the redle installed in the ocular of the microscope. Focus on the region or cell of interest.
Next, set up the light needed for visualizing the sample. Then give one or several pulses of laser light to the targeted region, depending on the desired degree of injury. Immediately after wounding, use a pipette to gently remove the embryo from the agros and place it back into egg water for recovery.
After recovery for the desired period of time, fix the embryo, perform immunohistochemistry, and analyze using confocal microscopy. Laser mediated injury was performed on immobilized one day old embryos Acton Stainings show that a few laser pulses can generate a small wound, easily recognizable by the damaged myofibrils that appear as acton coils. A higher number of laser pulses will result in massively damaged somites where most myo fibrils are destroyed after recovery for five hours.
Actin stainings show that myo fibrils within small injury sites have recovered and span the width of the so mite. We find that the speed of recovery depends on the size of the damaged tissue with small injuries healing faster than larger ones. Additional information can be gained by staining injured tissue with an antibody against SERP one, which is localized along recovering myo fis.
Within injured muscle cells at the site of laser injury indicated by a red bolt, the ablated cell has reduced GFP fluorescence. This figure demonstrates that after injury recovery phase and antibody staining, the target cell shown by the white arrow is recognized based on the pattern of mosaically labeled cells surrounding it as indicated by yellow arrows. The approach shown here allows one to follow over time how the laser mediated injury affects Semitic muscle cells.
Cells are recorded live to assess the immediate effects within the first minutes upon laser injury. Then time lapse recordings can be performed for any desired time interval to closely follow how cells rearrange around wounds. In this example of live imaging, the black arrow indicates where the laser injures the Semitic muscle at 30 HPF.
The ends of a damaged muscle cell are slowly retracting towards the so mite boundaries to the left and to the right of the black arrow. This method can be used in conjunction with other techniques such as Q-R-T-P-C-R or Western Blot in order to determine changes in gene expression or protein expression. But also time-lapse analysis can be conducted to investigate, for example, the immune response following organ injury.
So this method really has many possible applications.
