Analysis of Coronary Vessels in Cleared Embryonic Hearts

We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.

Transcript

The overall goal of this BABB base tissue clearing method is to allow non-invasive imaging of labeled structures deep within samples of interest. This method can help answer key questions in the field of cardiovascular development such as which cells or molecules contribute to normal coronary formation. The main advantages of this technique are that it is straightforward to perform doesn't require expensive reagents and it's quick compared to other tissue clearance methods.

The implications of this technique extend towards the therapy of coronary heart disease because understanding vessel formation may lead to therapeutic strategies for preventing disease or restoring normal cardiac function. On the day of the procedure use a dissecting microscope and forceps to carefully open the eutherian sacs of a timed pregnant mouse. Removing the yolk sac from each embryo and severing the umbilical cord.

To remove the heart, use stainless steel minutemen pins to fix each embryo on its back in a PBS filled silicon elastomer coasted 35 millimeter Petri dish. And use fine forceps to carefully open the chest and to sever the great vessel's interior to the heart. Reaching behind the heart with the forceps grasps the posterior cardiac vessels and pull gently to remove the organ.

Place the heart in a fresh 35 millimeter Petri dish containing PBS. Use fine forceps to trim away the lung tissue as necessary and transfer the heart into 1 well of a 48 well plate containing cold PBS. When all of the hearts have been collected use a fine-tipped plastic Pasteur pipette to replace the PBS in each well with 0.5 milliliters of 4%paraformaldehyde in a fume hood.

At the end of the fixation period remove the fixative and rinse the hearts two times in cold PBS. To permeabilize the hearts for whole mount immunostaining transfer the samples to 1.5 milliliter microcentrifuge tubes and rinse the tissues with three 10 minute washes in 0.5 to 1 milliliter of PBST with rotation at room temperature. After the third wash block the hearts for one hour with rotation at room temperature in 1 milliliter of freshly prepared 10 percent goat serum in PBST.

Then replace the serum with 400 to 500 microliters of the primary antibody of interest in fresh blocking buffer for an overnight rotation at four degrees Celsius. The next day remove the antibody and wash the hearts with six one hour rinses in one milliliter of PBST with rotation in room temperature. After the last wash replace the PBST with fresh blocking buffer continuing the appropriate fluorophore conjugated secondary antibody for an overnight incubation in four degrees Celsius protected from light.

Then wash the hearts six times with PBST as just demonstrated. To prepare silicon elastomer-coated dishes for confocal microscopy, in a fume hood place one cap from a four milliliter screw-top vial in the center of one optical glass-bottomed culture dish for each heart. Then, wearing safety glasses, fill each dish with silicon elastomer to an approximately 0.5 centimeter depth.

Using an applicator cartridge to mix the two components as they are applied according to the manufacturer's instructions. Allow the compound to cure for at least 24 hours making sure that the vial cap remains in the center of each dish. Removing the caps from the plates when the elastomer is set.

Before clearing the hearts, rotate the samples through a methanol-diluted in PBS series at room temperature in foil-covered 1.5 milliliter microcentrifuge tubes for five minutes each. After the 75 perfect methanol rotation, immerse the hearts in 100 percent methanol for three five minute rotations. After the last dehydration rotation, place hearts up to embryonic day 13.5 in the center of each elastomer well.

Orienting the samples dorsal-side up under the dissecting microscope. Use a fine-tipped plastic Pasteur pipette to remove any methanol from around each of the hearts. Then, in a fume hood, use a new pipette to add a 50 to 50 freshly prepared BABB to methanol solution in a sufficient volume to immerse the hearts.

Confirm the orientation while the hearts are still opaque. It is very important to orientate the sample according to the position of the confocal laser before clearing is complete to ensure good imaging of the coronary vessels or other structures of interest. After five minutes, transfer the solution to a glass waste bottle in the fume hood and add enough fresh BABB alone so that the samples sit within a drop of solution.

The samples should be clear within five minutes. When the tissues are clear, replace the solution with a minimal volume of BABB. The samples are now ready to image.

At embryonic day 11.5 PECAM1 antibody mainly stains the endocardial lining of the heart and outflow vessels. However a few endothelial cells can also be observed in the left ventricular wall. Here the increased vasculature approximal to the aorta can be observed in an embryonic day 12.5 heart.

This 12 slice z projection shows the whole parry trunkloplexus of an embryonic day 12.5 heart. However as some vessels are localized eventually to the heavily PECAM1 stained allumum of the aorta splitting the z stack into a number of sub-stacks provides more visual information. These five slice z projections of an embryonic day 15.5 heart facilitate visualization of the aortic and pulmonary valves by PECAM1 staining.

With the branches and interconnections of the coronary capillary network more clearly observed in the smaller sub-stack projections. Imaging software analysis allows manual segmentation of the samples to highlight the coronary arteries with or without the surrounding vasculature. The vasculature of whole embryos can also be examined using PECAM1 staining and BABB clearance.

Further staining of day 11.5 embryos with PECAM1 imaged immediately after BABB clearance or after approximately two months demonstrates that the fluorescent signal is strong enough to image after prolonged storage in the clearing solution. Once mastered, this technique can be completed in around a week from dissection to image capture. Whilst the clearing and mounting of small samples alone can completed in 30 minutes.

After watching this video you should have a good understanding of how to dissect, florescently label and BABB clear embryonic hearts to be ready for confocal microscopy imaging. Following this procedure, other methods like mulitphotonic imaging can be performed in order to answer additional questions, for examples relating to cellular morphology during coronary vessel formation. This technique should enable researchers in the field of developmental biology to explore blood vessel development in mouse embryonic hearts and other model systems.

While attempting this procedure it's important to remember to treat the samples gently to prevent damaging the delicate embryonic tissue. Don't forget that working wit BABB can be extremely hazardous. Under precautions such as wearing gloves, a lab coat and eye protection should always be taken while performing this procedure.

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Journal (Developmental Biology)

Journal (Developmental Biology)

Analysis of Coronary Vessels in Cleared Embryonic Hearts

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