Recovery of Adult Zebrafish Hearts for High-throughput Applications

The overall goal of this procedure is to quickly and reliably dissect adult zebrafish hearts for downstream applications. This is accomplished by first euthanizing adult zebrafish and fixing the fish if appropriate. Next, the fish is decapitated.

Then the dorsal aorta is cut and the fins with the underlying bones are removed. Finally, the heart is removed from the remaining tissue and placed in a destination buffer of choice. Ultimately, microscopy is used to show recovery of intact hearts for further analysis.

The main advantage of this technique over existing methods is that it reproducibly and quickly yields fully intact hearts. For analysis Before carrying out dissection, have the following buffers and solutions prepared. 500 milliliters of egg water, 200 milliliters of 0.03%trica in egg water, 100 milliliters of one X-P-B-S-R-B-C lysis, buffer, and a destination buffer of choice in a micro fuge tube on ice.

For the dissection setup. Begin by positioning a gooseneck light source over a dissecting microscope. Place the lid of a nine centimeter Petri dish on the dissecting microscope stage and pour a thin layer of one XPBS into it to wash the heart after dissection, place the bottom of the Petri dish with about 15 milliliters of one XPBS on the countertop.

Near the dissecting scope, place a razor blade, two pairs of forceps, optional micro scissors, and a transfer pipette Beside the microscope, prepare a container of ice if euthanasia by rapid cooling will be performed to euthanize using trica. Pour 200 milliliters of trica in egg water into a 250 milliliter glass speaker. Prepare a biohazard bag for disposal of zebrafish carcasses.

After euthanizing a fish by rapid cooling or trica, quickly pick up the fish by its tail fin and lay it on its side in the Petri dish cover filled with one XPBS. Use the forceps to lift a pectoral fin with one hand while using the razor blade in the other hand, position just posterior to the attachment of the pectoral. Fin to decapitate the fish while visualizing the fish head under the microscope.

Place a tying of a forceps into the mouth of the fish through the brain, and position the other ty outside the head across the eye to steady it with both tin of the forceps against the bottom of the Petri dish and the ventral surface of the head facing up. Use a second forceps to cut the ventral attachment of the operculum to the body. Next, slightly lift the tissue to expose the dorsal aorta underneath, which appears as a white structure with a red stripe of blood in the aortic lumen.

Then use forceps to cut the dorsal aorta by pinching the aorta and pulling upwards. Use the second forceps to grasp the pectoral fin from its base, including the body cartilage at the base of the fin, and lifted off carefully examining the fin to ensure that the heart hasn't come out with it. The heart should now be visible, still intact and connected to the remaining fish head.

Now release the fish head with the first forceps and use both forceps to tease the heart away from the pericardium before grasping the remainder of the dorsal aorta using forceps. Grasp the heart by the edge of the dorsal aorta or bulbous arteriosis and place it in the fresh dish of one XPBS, moving it back and forth in the buffer to wash away as much blood as possible. Alternatively, to remove all possible blood cells, use a dish of RBC lysis buffer to wash the heart in a similar manner.

If preserving the heart is not necessary for downstream applications, use forceps or micro scissors to open the heart cavity before rinsing away blood cells. If separate heart chambers are needed, dissect the atrium ventricle and bulbus arteriosis apart from each other. Finally, transfer the heart or separate chambers to their next buffer on ice.

Using the method described in this video, an adult zebrafish heart can be dissected in less than one minute and is reliably intact. Traditional methods can take over five minutes and require cutting blindly into the pericardium. This commonly causes damage or loss of the atrium or bulbous, arteriosis using the method described.Here.

Hearts maintain their structural integrity and are suitable for histology. For example, the outflow and atrial ventricular valves are identifiable in this section. In addition, as seen here, the hearts are suitable for electron microscopy as well as for applications where the heart is dissociated or pulverized.

This technique can be quickly mastered and is suitable for juvenile zebrafish older than one month, and for adults of all ages. After watching this video, you should have a good understanding of how to efficiently dissect adult zebrafish hearts.