Immunofluorescence Microscopy
Diane C. Shakes, David M. Miller III and Michael L. Nonet
Immunofluorescence microscopy is a powerful technique that is widely used by researchers to assess both the localization and endogenous expression levels of their favorite proteins. The application of this approach to C. elegans, however, requires special methods to overcome the diffusion barrier of a dense, collagen-based outer cuticle. This chapter outlines several alternative fixation and permeabilization strat- egies for overcoming this problem and for producing robust immunohistochemical staining of both whole animals and freeze-fractured samples. In addition, we provide an accounting of widely used antibody reagents available to the research commu- nity. We also describe several approaches aimed at reducing non-specific back- ground often associated with immunohistochemical studies. Finally, we discuss a variety of approaches to raise antisera directed against C. elegans antigens.