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The left ventricle was perfused intra-aortically with 250 ml of ice-cold, 0.1 M buffer phosphate [containing (mM): 137 NaCl, 2.7 KCl, 10 Na 2 HPO 4 , and 2 KH 2 PO 4 ] with heparin (10000 U/L), pH 7.4, at a flow rate of 18–25 ml/min, followed by 4% paraformaldehyde +12% picric acid solution until transparent liquid flowed from the right ventricle