N 3 dimethylaminopropyl n ethylcarbodiimide hydrochloride carbodiimide

Wild-type and Cd36^−/− mice were given 80 µl of [¹³C]fatty acids (about 1 mg/µl; Cambridge Isotope Laboratories) by gastric gavage. In other experiments, mice were anesthetized with ketamine/xylazine and given an intravenous injection of 200 µl of ²H-TRLs (40 µg triglycerides) through the inferior vena cava (for the 30-sec, 1.5-min, and 2-min time points) or the tail vein (for the 30-min time point). After the injection of ²H-TRLs, the diaphragm was cut to stop the heart at the indicated time points. Immediately thereafter, the mice were perfused with 10 ml of ice-cold PBS at 3 ml/min (5 ml though the right ventricle and 5 ml through the left ventricle). Next, mice were perfusion-fixed through the left ventricle with ice-cold 4% N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (“carbodiimide;” Sigma-Aldrich) (mass/vol) and 0.4% glutaraldehyde (Electron Microscopy Sciences) (vol/vol) in a 0.1 M phosphate buffer. The apex of the left ventricle was collected and cut into 1-mm³ pieces. The tissue was then placed in a 0.1 M phosphate buffer containing 4% carbodiimide and 2.5% glutaraldehyde overnight at 4°C.