Detection of Protein Aggregation in Neurodegenerative Diseases

Abstract

Protein aggregates/inclusions are pathological hallmarks of a wide spectrum of neurodegenerative ­diseases. These aggregates have different shapes, sizes, distribution, and protein composition, which are unique features used for pathological diagnosis. The aggregates per se are also used as molecular targets for designing therapeutic approaches. Detection of these aggregates is generally achieved by using immunostaining methods, most often by immunohistochemistry. In clinical and pathologic practice, the neurologic tissues to be examined are generally fixed with formalin and processed to paraffin-embedded tissue blocks. These treatments result in covalent cross-linking of the protein molecules and preserve the tissue morphology, but dramatically mask the antigens, making it often difficult to detect the aggregates. Therefore, removal of the cross-linking of antigens is a critical step for effective detection of these aggregates. In this chapter, we discuss and present immunostaining methods with a focus on the effectiveness of antigen-retrieval methods. In our experience, a treatment of tissues at 125°C for 20 min represents a relatively ideal antigen-retrieval method not only preserving the tissue morphology, but also providing efficient antigen retrieval. Using this method, we successfully detected some protein aggregates that escaped detection when other antigen-retrieval methods were employed.