Immunofluorescence microscopy.
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 strategies 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 community. We also describe several approaches aimed at reducing non-specific background often associated with immunohistochemical studies. Finally, we discuss a variety of approaches to raise antisera directed against C. elegans antigens.
Department of Biology, College of William and Mary, Williamsburg, Virginia, USA.
Diane C
DC
Shakes
David M
DM
Miller
Michael L
ML
Nonet
D000818:Animals; D000909:Antibodies, Helminth / Q000096:biosynthesis; D000947:Antigens, Helminth / Q000032:analysis; D002021:Buffers; D017173:Caenorhabditis elegans / Q000033:anatomy & histology / Q000196:embryology / Q000502:physiology*; D029742:Caenorhabditis elegans Proteins / Q000032:analysis* / Q000378:metabolism; D004625:Embryo, Nonmammalian; D047108:Embryonic Development; D005404:Fixatives; D005455:Fluorescent Antibody Technique; D005614:Freeze Fracturing; D007814:Larva / Q000033:anatomy & histology / Q000502:physiology*; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*; D016707:Tissue Fixation
D000909:Antibodies, Helminth; D000947:Antigens, Helminth; D002021:Buffers; D029742:Caenorhabditis elegans Proteins; D005404:Fixatives