A Method to Detect Cytochrome c Oxidase Activity and Mitochondrial Proteins in Oligodendrocytes.

Abstract

Cytochrome c oxidase or mitochondrial respiratory chain complex IV is where over 90% of oxygen is consumed. The relationship between complex IV activity and mitochondrial proteins, which provides a guide to understanding the mechanisms in primary mitochondrial disorders, has been determined by histochemistry (complex IV activity) and immunohistochemistry in serial sections. In the central nervous system (CNS), mitochondrial activity and immunoreactivity have been determined in populations of cells in serial sections as capturing cells in more than one section is difficult. In this report, we describe a method to determine complex IV activity in relation to mitochondrial proteins at a single-cell level in the CNS. We performed complex IV histochemistry and immunohistochemistry consecutively in snap-frozen sections. Although the product of complex IV histochemistry reduces the sensitivity of standard immunohistochemistry (secondary antibody and ABC method), the biotin-free Menapath polymer detection system enables mitochondrial proteins to be detected following complex IV histochemistry. The co-occurring chromogens may then be separately visualized and analyzed using multispectral imaging. Our technique is applicable for exploring mitochondrial defects within single cells, including oligodendrocytes, in a variety of CNS disorders and animal models of those diseases.