Immunofluorescence microscopy for DIGE-based proteomics

Abstract

Alterations in the proteome of a tissue in different settings, as assessed by difference gel electrophoresis, can be verified for single proteins using immunohistochemistry. In fluorescence immunohistochemistry, an antibody to a particular antigen is applied to tissue sections, and fluorophores conjugated to a secondary antibody allow for the detection of target antigen with fluorescent microscopy. Visual comparison is sufficient for the detection of significant alterations in the abundance of a certain protein in different settings. Additionally, unlike large-scale proteome analyses and Western blot methods, expression of target protein can be analyzed at the cellular level by immunohistochemistry. In this chapter, a protocol for the application of fluorescence immunohistochemistry for the detection of dystrophin in skeletal muscle sections is outlined, including sample preparation, tissue sectioning, and immunostaining.