Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The overall goal of this method is to quantitatively assess cardiac progenitor cell localization and distribution during heart organogenesis. This method can help answer key answers in the heart development field by allowing us to observe the localization of cardiac progenitor populations during normal embryonic development. The main advantages of this technique are that the volumetric imagery can yield quantitative information about tissue level dynamics and that the analysis pipeline can be automated for improved reproducibility.

Though this method provides information about early cardiac organogenesis, it can also be applied to the general study of early embryogenesis or to the morphogenesis of other organs of interest. On the morning of embryonic day eight, disinfect the pregnant mouse abdomen with 70%ethanol and make an abdominal incision through both the skin and body wall. Beginning above one oviduct, carefully dissect the fat along the side of the uterus down to the cervix.

Make an incision through the cervix and continue to the upper end of the other oviduct cutting to release the entire uterus. Wash the excess blood from the uterus in a 10 centimeter dish containing fresh PBS and cut the mesometrium between each deciduom. Transfer the embryos to a six centimeter dish containing fresh PBS under a dissecting microscope and use number five fine forceps to remove the uterine tissue from the decidual tissue.

Next, carefully slice the tip of the embryonic half of the deciduom to reveal the embryos, pinching the deciduom to carefully push and pull the first embryo out. Dissect away as much extra embryonic tissue as possible without damaging the morphology of the animal and use a transfer pipette to place the embryo into a 1.5 milliliter tube containing fresh PBS on ice. When all of the embryos have been harvested, aspirate the PBS from each tube and rinse the embryos with fresh PBS.

After thoroughly aspirating the PBS from each tube, fix the embryos in 4%paraformaldehyde for one hour at room temperature, followed by three washes with fresh PBS, then store the embryos at four degrees Celsius. Begin by replacing the PBS with one milliliter of blocking buffer for a four-hour incubation at room temperature. At the end of the incubation, replace the blocking buffer with the appropriate primary antibody mixture and diluted blocking buffer for an overnight incubation at four degrees Celsius.

The next morning, aspirate the excess antibody and wash the embryos three times for one hour per wash in 0.1%Triton in PBS. After the third wash, add the appropriate secondary antibody mixture to the embryos for a three-hour incubation at room temperature, followed by three washes as just demonstrated. Next, counterstain the embryos with DAPI in PBS for 10 minutes followed by two five-minute washes, then slowly suspend the embryos in anti-fade mounting medium allowing the embryos to equilibrate for at least one hour with periodic gentle flicking to help the specimens drop into the solution.

Before mounting the embryos, make two parallel stacks of five to six layers of double stick tape 15 to 20 millimeters apart on one microscope slide per embryo and place a 15 microliter drop of anti-fade medium onto each slide between the tape stacks. Carefully transfer one embryo into each drop and use a dissection microscope and fine forceps to position the embryos with their anterior sides facing away from the slide and with their body axis in line with the long axis of the slides. To place the coverslips, first rest one side of the glass on one stack of double stick tape and use forceps to gently lower the coverslip until it contacts the other tape stack.

It’s particularly important to carefully position and mount the embryos. Embryos that aren’t positioned correctly will be much more difficult to image and may require additional processing to get good quantifiable data. Use a pipette to add additional anti-fade medium between the slide and the coverslip to keep the sample from drying out during the imaging and select the highest magnification objective available that allows the whole cardiac crescent region to be captured in one field of view.

Then set up the imaging parameters using standard best imaging practices and use Nyquist sampling rates to determine the XYZ voxel dimensions of each embryo. To analyze the embryo images, load the raw image data sets into the appropriate image analysis software and open the first file in the surpass view. The data will load in a 3D view in the maximum intensity projection mode.

Open the display adjustment window and select the reference channel only. Adjust the threshold of the image as necessary. If the signal to noise ratio is too low for segmentation, adjust the gamma.

Following best practices during image acquisition is critical for obtaining suitable data and will depend on the specific microscopy setup used. If adjustments to the gamma are needed, these images should not be used for comparing fluorescence intensities. Use the surface algorithm dialogue to create a new surface for the reference channel and check the segment only origin of interest box.

Adjust the bounding box region so that it contains the region of interest as delineated by the reference stain and select the reference channel from the source channel dropdown menu. Set the smoothing to half of the Z voxel size and use the threshold sliders to adjust the lower limit to its absolute intensity to ensure that the surface does not extend beyond the channel signal. Filter the objects by voxel number or volume rejecting any background objects that the algorithm might have generated and finish the algorithm.

Next, select the newly created surface and choose the mask set function from the edit window. Select the experimental channel of interest taking care that the duplicate channel before applying mask checkbox is selected and select the mask channel only in the display adjustment window. Then create a new surface as just demonstrated.

To obtain the volumetric data with each surface selected, open the detail sub tab in the statistics tab and select average values from the dropdown menu. The total volume of the surface will be found in the sum column of the generated table. Here, an embryonic day 8.25 mouse embryo with GFP positive ventricular cardiovascular progenitor cells labeled with an anti-cardiac crescent antibody as just demonstrated is shown.

After selecting the reference channel for the stained tissue, the intensity and gamma are adjusted before beginning the create surfaces algorithm. The region of interest is selected and the level of surface detail is chosen. The initial surface is thresholded to include all true signal in the surface.

Filtering is then performed to remove small background fragments to yield a final reference surface. By selecting the reference surface, the comparison channel can be duplicated and masked. The intensity and gamma for this channel are adjusted before generating a second surface through the same sequence of steps.

The total volume for each surface is then calculated automatically and can be compared both quantitatively and visually. While attempting this procedure, it’s important to remember that the quality of the dissected embryos, the antibody efficiency, the mounting orientation, and the imaging setup all affect the quality of the final data. In addition to this quantitative analysis pipeline, other methods such as deconvolution can be performed to yield additional qualitative data.

After its development, this technology paved the way for researchers in cardiac development to explore the dynamics of novel cardiac progenitor populations during early embryogenesis.