Chapter 10 - Histochemical and immunohistochemical staining methods to visualize mitochondrial proteins and activity

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Abstract

We describe here reliable histochemical and immunohistochemical techniques to visualize mitochondria and respiratory chain dysfunction in tissue sections. These morphological methods have been widely used for years, and yet remain relevant to obtain insight into the pathogenesis of mitochondrial diseases. Today, mitochondrial medicine is changing rapidly and genetic information plays an increasing role in the diagnostic process, owing to advances in next-generation sequencing. However, tissue analysis and morphological categorization remain essential, especially when genetic abnormalities of unknown significance might complicate a diagnostic odyssey. Furthermore, tissue assessment is an essential step in laboratory investigation using animal or cell models, in order to assess the distribution, severity, and/or progression of the disease, and to evaluate the effects of possible treatments. Optimized and reproducible staining and imaging methodology are the key elements for accurate tissue assessment. When these methods are used properly and integrated with wisely chosen genetic and biochemical approaches, powerful information can be obtained about the structure and function of mitochondria in both animal model systems and human patients. While the described protocols refer to skeletal muscle and brain mitochondria, the methods described can be applied to any tissue type.

Section snippets

Rationale

One of the first histochemical methods to detect alterations of mitochondria in skeletal muscle was the modified Gomori trichrome method of Engel and Cunningham (1963). In this method, frozen sections (6–10 μm thick) are incubated in Gill's hematoxylin for 30 min, rinsed, placed in Gomori trichrome solution for 1 h, and dipped in 0.2% acetic acid, dehydrated in graded ethanols, and cleared in xylene or xylene-substitute clearing agent to be eventually mounted with Permount. This stain results in

Rationale

Immunohistochemistry is a technique based on the detection of epitopes using antibodies, allowing for the identification of specific proteins in single cells. Immunohistochemical techniques can be used with frozen tissue or formalin-fixed paraffin-embedded tissue, which represents an important advantage compared to histochemistry techniques. Moreover, there is a great array of commercially available antibodies against both mtDNA- and nDNA-encoded subunits of the respiratory chain complex, other

Acknowledgments

Images were collected in the Confocal and Specialized Microscopy Shared Resource of the Herbert Irving Comprehensive Cancer Center at Columbia University, supported by NIH grant #P30 CA013696 (National Cancer Institute). The confocal microscope was purchased with NIH grant #S10 RR025686.

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