Visualization and Analysis of Pharyngeal Arch Arteries using Whole-mount Immunohistochemistry and 3D Reconstruction

This method allows each pharyngeal arch to be compartmentalized. Endothelial cell numbers can then be quantified in each arch as a means to study pharyngeal arch artery development. The main advantages of this approach are the ability to visualize the relationship among different vascular structures and the ability to quantitatively determine how the structures affected the mutants.

Aortic arch arteries are commonly mutated in congenital heart disease. Our method allows the study of how mutations alter the physiological process of vessel formation and remodeling. Although we use this method to gain insights into the mechanisms of congenital heart disease, it can be applied to other systems particularly with the advent of light-sheet microscopy.

Begin by using a glass pipette to transfer each mouse embryonic day 9.5 or 10.5 embryo into individual two milliliter tubes containing one milliliter of PBS. After fixation, use fine forceps to carefully pinch the embryo just above the hindlimb and make a transverse cut to remove the posterior half of the embryo. To permeabilize the embryos, replace the PBS with PBST and place the embryos at four degrees Celsius with gentle agitation overnight.

The next day, replace the PBST with 600 microliters of blocking buffer without touching the embryos for a 16 to 18-hour incubation at four degrees Celsius with gentle agitation. The next morning, replace the blocking buffer with 600 microliters of the primary antibody of interest per tube for a four to five-day incubation at four degrees Celsius with gentle agitation. At the end of the incubation, wash the embryos in one milliliter of PBST for four to five hours with gentle agitation at room temperature changing the PBS every hour, followed by an overnight incubation in fresh PBST at four degrees Celsius and four to five additional one-hour washes the next day.

After the last wash, replace the PBST in each tube with 600 microliters of the appropriate secondary antibody solution for a four to five-day incubation at four degrees Celsius. At the end of the incubation, wash the embryos in one milliliter of fresh PBST per wash as just demonstrated and use a glass pipette to gently transfer each embryo into individual plastic paraffin molds. Carefully remove any PBST from around each embryo and orient each embryo in a sagittal position.

Then quickly add about 500 microliters of hot agarose to each mold until each embryo is just covered and place the molds on ice covered with aluminum foil until the agarose has solidified. For dehydration of the embryonic samples, use a clean scalpel to cut a block of agarose around each embryo and use forceps to grasp the agarose for transfer into a labeled two milliliter tube containing one milliliter of 25%methanol. After a one-hour incubation with gentle agitation in the dark, replace 25%methanol with one milliliter of 50%methanol per tube for an additional one-hour incubation in the dark.

After the end of the dehydration, replace the 100%methanol with one milliliter of 50%BABB for a one-hour incubation with gentle agitation in the dark. At the end of the incubation, replace the 50%BABB with one milliliter of 100%BABB per tube for a one-hour incubation with gentle agitation in the dark, followed by a second incubation with 100%BABB as just demonstrated. To mount the embryos for imaging, remove the adhesive from a fast well bumper and place the bumper onto a 24 by six milliliter number 1.5 glass coverslip.

Apply gentle pressure to the adhesive to remove any air bubbles between the coverslip and the bumper and carefully discard the 100%BABB from each tube. Then use fine forceps to carefully transfer the embryo into the fast well without touching the embryo and place a second coverslip onto the bumper. To surface the endothelium in the entire third pharyngeal arch, open the image of interest in the software and click add new surface.

Double-click surface one and rename the new surface to third pharyngeal arch. Select skip automatic creation, edit manually, and set the surface orientation to the YZ plane. Use the slice position to place the third pharyngeal arch surface plane to where the third PAA and dorsal aorta connect.

Rotate the image so that the third pharyngeal arch surface plane is in view and turn off ortho slicer. Under draw contour mode, select the distance drawing mode function and adjust the parameter settings as necessary. When all of the parameters have been set, press the Escape key and click draw to trace the perimeter of the third pharyngeal arch with the mouse cursor.

Then use the slice position to move 10 to 25 slices and trace the perimeter of the pharyngeal arch. When the entire arch has been traced, click create surface to generate the surface of the traced region. For masking of the surfaced structures, in the edit menu, select mask selection for the third PAA and DAPI and click OK.Then rename the new channel as PAA DAPI.

Repeat this step for the remaining channels. To visualize the endothelial plexus separately from the PAA, select mask selection for the third PAA and select DAPI. Uncheck select voxels outside surface to and check select voxels inside surface to.

Set select voxels inside surface to to zero and click OK.Rename the new channel as non-PAA DAPI. Repeat this step for the remaining channels. Select mask selection for the third PA and select non-PAA DAPI and click OK.Rename the new channel as plexus DAPI.

Repeat this step for the remaining channels. To quantify the individual endothelial cells within each structure of interest, in the display adjustments panel, turn off all of the channels except the PAA ERG channel. Under the properties menu, click add new spots and click to rename spots one PAA total number of endothelial cells.

Click the blue arrow and select the PAA ERG channel for the source channel. Adjust the estimated XY diameter to four micrometers and click the blue arrow to proceed to the next panel. Use the sliding scale to adjust the number of spots to ensure that each ERG positive endothelial cell nucleus is represented by one spot.

Next, click the green double arrow and turn off the PAA ERG channel in the display adjustment panel. Turn on the PAA VEGFR2 channel to visualize the PAA endothelium and click edit and add/delete to select surface of object. Then press Escape and hold down Shift to delete any spots that are not VEGFR2 positive and click the statistics tab to determine the total number of endothelial cells.

Whole mount immunofluorescence produces clear and clean results allowing the 3D reconstruction of the pharyngeal arch endothelium. In this image, the presence of the large bright dots is the result of particulate in either the antibody or blocking buffer solutions. After pharyngeal arch and PAA surface staining, use of the mask function allows the surfaced regions to be visually separated and independently analyzed.

Masking also allows the individual analysis and quantification of endothelial cell numbers within each structure. For example, here the spot feature was used to quantify the total number of endothelial cells in both the PAA and the plexus by assigning a single spot for each nucleus expressing ERG. In this image, an ERG positive VEGFR2 negative spot that has been generated by the spot function can be observed.

Therefore, it is essential to verify that each dot recognized by the software actually represents a single endothelial cell. To obtain clean and clear images, remember to spin down all the solutions and to properly and thoroughly wash each embryo after antibody incubations. It is important to remember that BABB is corrosive and toxic.

Handle and discard the solvent properly and be sure to seal the melted embryos completely to prevent BABB leakage.