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Analysis of Cytochrome c Release by Immunocytochemistry.

Cytochrome c is normally localized between the inner and outer membranes of mitochondria in healthy cells. However, during apoptosis, it is released into the cytoplasm, where it binds to apoptotic protease activating factor. Caspase-9 is then recruited and activated by this complex in a process known as the induced proximity model. Release of cytochrome c from mitochondria is therefore a critical event in apoptosis and various protocols are available for its measurement. Cytochrome c in mitochondria has a punctate localization pattern in the cell and its translocation to the cytoplasm results in a diffuse distribution. This is visually striking and easily observed by immunocytochemistry. This protocol describes the use of immunocytochemistry to assay cytochrome c release during apoptosis.