Preparation and Immunolabeling of Caenorhabditis elegans
Adapted from Basic Methods in Microscopy (eds. Spector et al.). CSHL Press, Cold Spring Harbor, NY, USA, 2006.INTRODUCTION
The use of antibodies to visualize the distribution and subcellular localization of gene products powerfully complements genetic and molecular analysis of gene function in Caenorhabditis elegans. The challenge to immunolabeling C. elegans is finding the fixation and permeabilization methods that effectively make antigens accessible without destroying the tissue morphology or the antigen. Embryos are surrounded by a chitinous eggshell and larvae and adults are surrounded by a collagenous cuticle, each of which must be permeabilized to allow penetration of antibodies. In addition, antigens and antibodies are sensitive to different fixing and permeabilizing conditions. This protocol describes two methods for tissue fixation. The whole-mount freeze-cracking method is a good starting point as it is easy and works well with most antibodies and with embryos, larvae, and adults. In the tissue extrusion method, gonads and intestines, which are extruded from the carcass, are well fixed and permeabilized. Tissues remaining in the carcass are not usually stained well. The protocol concludes with an antibody incubation procedure in which fixed worms are incubated overnight with primary antibody, subsequently exposed to secondary antibody, and mounted for viewing.
