The overall goal of the following experiment is to perform fate mapping and lineage tracing of selected single cells in zebrafish embryos. This is achieved by chemically conjugating caged fluorescein to high molecular weight dextran. Next, the prepared caged fluorescein dextran is micro injected into zebrafish embryos, which is then photo activated in selected cells using two photon absorption.
After photo activation of the embryos, immunohistochemical analysis is performed to detect the uncaged fluorescein dextran in order to fate map the activated cells. Ultimately, this method can be used to determine the lineage contribution and tissue localization of the photo activated single cells. The main advantage of this technique over existing methods, such as standard fluorescence microscopy, is that it can be used to differentially label single cells for faith mapping.
This method can help us answer key questions in developmental biology such as how undifferentiated precursor cells can contribute to different tissue types, Visual demonstration is critical since the embryo mounting and photo activation steps are difficult to learn based on text description alone. Before getting started, synthesize caged fluorescein dextran and store it in metal foil covered tubes. Then micro inject embryos at the one to two cell stage with the synthesized cage fluorescein dextran.
After the embryos have reached the appropriate stage of development, coate them. Then mount the coated embryos in low melting aeros For photo activation. When the arose has solidified load the LSM five 10 software.
Place a transparent yellow filter in the white light beam path and then select scan new image and click start expert mode. Select the laser tab, turn on the argon iron and the mi tai laser sources. Set the mi tai wavelength to seven 40 nanometers.
Next, select the config tab. Set the optical path configurations for the argonne ion and the mi tai laser. Then select the scan tab.
Change the objective to 20 times 0.8. Set the max scan speed by clicking max set mode method and number to line mean and one respectively under the channels tab deselect all laser sources except for the mi tai. Next, place a power meter in the optical beam path, and then select the con button to activate continuous scanning.
Adjust the transmission percent until the power meter reads an average laser power of 47 milliwatts. Then stop the scan by selecting the stop button under the channels tab. Deselect the mi tai laser source and select the argonne iron.
Then select the fast XY button again under the channels tab, adjust the pinhole. Detect gain, amplify a offset and amplify again for the Argonne iron laser to achieve the best image quality. Using the stage controller, find and focus on the cell to activate, then click the stop button Using the crop tool, crop the activated cell so that the crop factor under the mode tab displays 50 to 70.
Then stop the scan by selecting the stop button under the channels tab. Deselect the argonne ion laser and select the My Tai laser under the mode tab. Set the scan speed to 31.46 seconds.
Then select the single button to start the scan for the two photon activation of the caged fluorescein dextran of the selected cell. After activation, go to the channels tab and now deselect the mi tai laser and reselect the argon ion laser. Then under mode, set the zoom factor to one.Next.
Under the speed heading, click the max button to set the fastest scan speed. To review the photo activated region, select fast xy. Then select the channels tab and adjust the pinhole and detect again, check that the photo activated region is not laser ablated.
After preparing the photo activated embryos for uncaged fluorescein dextran immuno detection by following the protocol outlined in the accompanying manuscript. Wash the embryos six times in PBT and two times in AP buffer. Then transfer the embryos to nine well plates in AP buffer.
Next, aspirate the AP buffer and add N-B-T-B-C-I-P. Stop the N-B-T-B-C-I-P stain development by aspirating the N-B-T-B-C-I-P solution. Then wash the embryos in PBT.
Now recover the embryos and shake them on a rotating platform for five minutes of room temperature. Then repeat the washing PBT two more times. Finally, mount the embryos in 0.6%low melting aros and acquire images of the photo activated cells using an upright compound microscope.
In this figure, a brightfield image shown on the left and a fluorescent image shown on the right of a caged fluorescein dex strand micro injected zebrafish embryo before photo activation. As shown here, the embryo has been photo activated in one of the somites indicated by the arrow in the brightfield figure on the left with a wavelength of seven 40 nanometers, a scan time of 31 seconds, and an average laser power of 47 milliwatts. In the figure on the right post photo activation fluorescence from the uncaged fluorescein dextran can be observed in this figure.
Immune staining of uncaged fluorescein dextran is depicted a small region in the lateral plate, mesoderm of a 10. So mite stage embryo was photo activated using the same laser parameters as in the previous figure using the immuno detection procedure. The activated region as indicated here by the arrow, can be identified with minimal background staining Following this procedure.
Additional analysis such as immuno staining for different tissue specific markers can be performed in order to better characterize the location and fate of labeled cells. Using this technique, it paves the way for researchers in the field of vascular biology to study the origin of arterial in venous progenitor cells in developing zebrafish embryos. Don't forget that when working with chemical reagents such as NBT and BCIP, they can be extremely hazardous and gloves should always be worn.
