Integrated SDH, COX histochemistry, and MHC fibre typing in skeletal muscles: Methods and interpretation.

Skeletal muscle plays a vital role in metabolic homeostasis, accounting for the majority of glucose uptake, lipid oxidation, and adaptive thermogenesis. Its plasticity enables rapid, controlled remodelling in response to exercise, nutrients, hormonal changes, ageing, and disease. This metabolic plasticity is due to fibre-type heterogeneity. Each muscle fibre has distinct contractile and bioenergetic properties. Sirtuins are known critical regulators of skeletal muscle mitochondrial content and oxidative metabolism. Sirtuins, are NAD+-dependent acetylases and deacetylases that regulate mitochondrial biogenesis, redox balance, and cellular response to stress. Thus, studying the role of sirtuins in muscle physiology requires assays that can identify metabolic and contractile phenotypes. In this chapter, we provide a comprehensive histochemistry protocol for succinate dehydrogenase (SDH) and cytochrome c oxidase (COX) to assess mitochondrial oxidative capacity, and Myosin Heavy Chain (MHC) immunohistochemistry to assess fibre-type classification. Additionally, we have discussed detailed guidance for troubleshooting the critical steps of the protocol, including cryoinjury, tissue sectioning, staining optimisation, and imaging.