Detection of p62 on Paraffin Sections by Immunohistochemistry.

The study of autophagy in human disease is a rapidly expanding field. Diagnostic paraffin sections of a variety of patient tissues, including bone marrow, are available to researchers-yet are unsuitable for traditional autophagy quantification methods such as western blot or electron microscopy. This protocol outlines the immunohistochemical detection of the protein p62 (sequestosome-1, encoded by the gene SQSTM1)-an indicator of autophagic degradative activity-in slide-mounted paraffin sections such as bone marrow samples cut by a trephine. The p62 protein is an autophagic cargo adaptor, capable of binding to ubiquitylated proteins as well as autophagosome membrane proteins (LC3B and GABA(A) receptor-associated protein [GABARAP] family members) and hypothesized thus to target protein aggregates for lysosomal degradation. p62 itself is degraded by autophagy, remaining at low levels when autophagy is induced, and has been shown to accumulate when autophagy is deficient. Qualitative assessment and comparison of p62 staining between healthy and disease sections or disease subtypes will help target further investigation into the potential roles for autophagy in a variety of disorders.