1. Measuring fish and dissection

1. Place single fish into petri dish half filled with fresh PBS and lay it on its right side.
2. Measure length of fish from snout to base of tail (the caudal peduncle), not including tail fins, using a digital mm caliper (Figure 1). This is the standard length (SL).
3. Each fish can be assigned a number/letter combination to allow an individual fish and the resulting dissected heart to be tracked for later analysis. Data, including parental stock number, age at fixation, SL measurements, were entered into a spreadsheet and organized based on these labels.
4. Before dissection, label PCR tube strips with assigned tracking number and fill with PBT or other solution appropriate for further analysis.
5. Orient the fish ventral side up in petri dish while stabilizing the body with forceps holding the head between the eyes and gills (Figure 1).

Smaller fish (smaller than 12mm SL):

6. a) Using sharp forceps, or a micro-needle 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 PBT that move the fish. This is particularly problematic for smaller fish, thus a micro-needle can be used to remove pectoral muscles and fins and open the body cavity.
7. a) Gently use the forceps to scoop the heart out of the cavity from under the atrium. Alternately, in some fish you may be able to remove the heart by gently pulling the bulbous out and the rest of the heart will follow. Be careful of the location of the atrium if using this technique as it can easily be damaged. If there is extra tissue that comes with the heart, that is fine and can be cleared after.
8. a) If the fish contains a fluorescent cardiac marker such as Tg(myl7:GFP)^twu34, use fluorescent scope for dissection and verify removal of the heart by florescence (Figure 2).
9. a) Once the heart is removed from the cavity, use the forceps to hold the heart and a micro-needle to remove extra non-cardiac tissue and the epicardial lining from the outside of the heart.

Larger fish (greater than 12mm SL):

6. b) Using spring handled micro-scissors, make three incisions of less than 1mm deep: 1- transverse cut through the gills 2- transverse cut at anterior belly 3- sagittal cut on ventral side connecting both cuts (Figure 3).
7. b) 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 (Figure 4A). Remove this pericardial tissue and the heart will become visible (Figure 4B).
8. b) Use the micro-scissors to cut the artery connected to the bulbous arteriosus, located superior to the heart. Once this artery is cut, put the forceps tips under the atrium, use the forceps to scoop the heart out of the cavity. If there is extra tissue that comes with the heart, that is fine and can be removed later.
9. b) Once the heart is removed from the cavity, use the two forceps to hold the heart and remove extra tissue, or one pair of forceps to hold the heart and a micro-needle to remove extra, non-cardiac tissue. The epicardial lining, evident by its black pigmentation, can be removed from the outside of the heart if desired by gently scraping the tissue.