Table 2.

Troubleshooting.

Step	Problems	Causes	Suggestions
5,7	Sedation depth insufficient	Insufficient drug concentration for the individual	Have the mouse breath isoflurane through mask until depth of narcosis is sufficient.
7	Intubation failure	Difficulties locating larynx	Place cold light strictly in front of the larynx, perform second attempt or have a second operator perform intubation. If mouse is without ventilation and spontaneous breath for more than 1 min, consider killing the mouse.
7	Ventilation failure	Rupture of the lung, tension pneumothorax	Kill the mouse, check ventilation system, e.g., using an artificial lung, decrease tidal volume and ventilation pressures.
18–20	Bleeding into pericardium	Surgical manipulation on fragile structures	Kill the mouse.
20	Difficulties locating coronary artery	The coronary anatomy varies in strains	Gently push the heart out from the thorax by pushing against the right sight of the thorax. Slightly increase thoracotomy length to the right sight while avoiding pneumothoraces. In some strains, e.g., lacking endothelial NO synthase, visual location of the coronary artery is particularly difficult. In these cases, the typical location of the coronary could be assumed and the ligation performed. Ligation success will be obtained after staining for AAR and infarction-to-risk area.
28,31	Large remaining amount of blood in the excised heart	Insufficient anticoagulation	Add heparin (e.g., 5000–10000 IE per 10 ml preparation buffer) to the buffer in the cooling plate.
31	Difficulties in aortic cannulation	Short aorta	Aorta should be at a sufficient length. Do no resect below the aortic valve.
31	Leakage from the cannulated aorta	Insufficient ligation	Apply a second ligation quickly. Bear in mind that the total preparation time should stay limited to avoid further global hypoxia/ischemia. Omit the heart if leakage cannot be located.
32	Coronary ligation cannot be re-located	Specific anatomical characteristics in particular mouse strain	Leave the primary suture unligated in the thorax during in vivo reperfusion period. After heart excision re-ligates this suture.
52	Arrhythmia	Incorrect composition of perfusion buffer or long heart preparation time	Check pacemaker set-up and buffer composition. Keep track of your preparation time (until cannulation). Try to further minimize preparation time.