Biochemical and morphological detection of inclusion bodies in autophagy-deficient mice.

Autophagy-deficient mice exhibit the formation of ubiquitin-inclusions in the liver and brain, which is not attributed to the dysfunction of the ubiquitin-proteasome system. Moreover, it is also clear that a multifunctional protein p62/A170/SQSTM1 (hereafter referred to as p62) links autophagy and inclusion formation, being one of the key components of the ubiquitin inclusions. The ubiquitin/p62 inclusions can be detected in the detergent-insoluble fraction by western blot analysis, while morphological information can be obtained by immunohistochemistry at both the light and electron microscopy levels. Importantly, p62 has become a reliable marker, with which we can identify inclusions and estimate autophagic activity in diseased tissues or cells. In this chapter, we describe the methods used for biochemical and morphological detection of ubiquitin/p62-inclusions in autophagy-suppressed Atg7-deficient mice. These methods are suitable for examination of cells and tissues with conditions associated with reduced autophagy (e.g., aging and mice models of intractable diseases such as Alzheimer's disease), and their applications should enhance our understanding of the pathophysiological mechanisms involved in the formation of intracellular inclusions.