The overall goal of this procedure is to remove the heart from the zebra fish. This is accomplished by first collecting and fixing zebra fish. Next, the body cavity is opened to visualize the heart.
Then the heart is removed and cleaned of non-cardiac tissue. Finally, the heart is photographed. Ultimately, the morphology of the heart over time can be seen through microscopy, sectioning, and photography.
Visual demonstration of this method is critical as the keyto dissection. Sta are difficult to learn because zebrafish are small and the tissues are not distinct in color and can be hard to identify To generate zebra fish from the larvae through adult stages. For these experiments, perform individual pear or group matings with adult fish.
Clean the embryos and store them in a 28 degrees Celsius incubator. At five days post fertilization, separate fish into tanks at a 10 to 15 fish density and place them into the fish facility. Remove the fish from its tank at the appropriate age and place it in 0.2%Trica to anesthetize.
Place the anesthetized fish in ice water for 15 minutes to euthanize. Meanwhile, add 4%para formaldehyde or PFA in one XPBS to a dish. Incubate collected fish in the 4%PFA for one to two hours at room temperature, then overnight at four degrees Celsius in a Petri dish wrapped in perfil.
This keeps the fish flat and makes dissection much easier after fixation. Rinse twice in PBS with 0.1%Tween or PBT fish can be kept in PBT at four degrees Celsius for up to 10 days. Place a single fish into a Petri dish, half filled with fresh PBS and lay it on its right side.
Measure the length of the fish from snout to the base of the tail, not including the tail fins using a digital millimeter caliper. This is the standard length or sl. Assign each fish a number letter combination to allow an individual fish and the resulting dissected heart to be tracked for later analysis.
Enter all data, including all measurements into a spreadsheet and organized based on these labels before dissection. Label PCR tube strips with assigned tracking number and fill tubes with PVT or other solution appropriate for further analysis. Orient the fish ventral site up in a Petri dish while stabilizing the body with forceps, holding the head between the eyes and gills for fish shorter than 12 millimeters in standard length.
Using sharp forceps or a microneedle in a pin holder. Remove the pectoral muscles and fins from the body to reveal the heart. The constant motion of the forceps during dissection can cause currents in the PPT that move the fish.
This is particularly problematic for smaller fish. Thus, a microneedle needs to be used to remove pectoral muscles and fin and open the body cavity. Use the forceps to gently scoop the heart out of the cavity from under the atrium.
If the fish contains a fluorescent cardiac marker, use a fluorescent scope for the dissection and verify removal of the heart by fluorescence. Once the heart is removed from the cavity, use the forceps to hold the heart and a microneedle to remove extra noncardiac tissue and the epicardial lining from the outside of the heart. For fish longer than 12 millimeters in standard length.
Using spring handled micro scissors, make three incisions. First, make a transverse cut through the gills. Second, make another transverse cut at the anterior belly.
And third, make a sagittal cut on the ventral side connecting both transverse cuts. Using sharp forceps, remove the pectoral muscles and fins from the body to open the body cavity and reveal the silvery tissue of the pericardium. Remove this pericardial tissue and the heart will become visible.
Use the micro scissors to cut the artery connected to the bulbus arteriosis located superior to the heart. Once this artery is cut, place the tips of the forceps under the atrium and use the forceps to scoop the heart out of the cavity. If there is extra tissue that comes with the heart, that is fine as it can be removed later.
Alternatively, in some fish, it is possible to remove the heart by gently pulling the bulbus out and the rest of the heart will follow. Be careful of the location of the atrium if using the alternative technique as it can easily be damaged. If there's extra tissue that comes with the heart, this is fine and can be cleared after.
Once the heart is removed from the cavity, use the two forceps to hold the heart and remove the extra tissue or one pair of forceps to hold the heart and a microneedle to remove extra non-cardiac tissue. To photograph the zebrafish heart, prepare a 23 gram per liter nutrient agar petri dish and cover the solidified agar with PBS. Use the forceps to make a well in the agar that allows the heart to fit in the proper orientation.
Remove the heart from the PCR tube by holding the tip of the bulbus. With the forceps firmly. Place the heart in the agar dish.
Orient the heart in the agar to photograph. The sample can be rotated after each picture to all possible orientations. Use overhead, light, and short exposure times to photograph the hearts.
Make adjustments for light quality using the exposure as necessary to obtain the best photograph. An example of a well dissected clean heart is shown here. Each heart will have some differences in overall morphology, but the heart should be complete containing an intact ventricle atrium and bulbous arteriosis after its development.
This technique paved the way for researchers in the field of zebrafish cardiac development to explore cardiac maturation in various stages of zebrafish development.
