Immunohistochemical Determination of Cytosolic Cytochrome c Concentration in Cardiomyocytes

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Abstract
Cytochrome c release from the intermembrane space of mitochondria is one of the triggers of apoptosis. There is no histochemical method available to demonstrate cytochrome c in cryostat sections, possibly because small cytosolic proteins diffuse readily into aqueous fixation media. This report shows that it is possible to demonstrate cytochrome c release in cardiomyocytes in failing myocardium using vapor fixation of cryostat sections and immunohistochemistry. The method is calibrated using sections from gelatin blocks containing known concentrations of cytochrome c. The method is applied to the hypertrophied right ventricular wall of rats in which pulmonary hypertension was induced by monocrotaline. Cytochrome c release is found in a fraction of the cardiomyocytes, leading to a mosaic-staining pattern. Cytochrome c release was found in myocytes over the full range of cross-sectional area (from 1 to 3.9 times control) in the hypertrophied myocardium. Cytosolic cytochrome c concentrations up to 0.4–0.5 mM occur frequently.

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