Immuno-EM localization of GFP-tagged yolk proteins in C-elegans using microwave fixation

Because of the presence of a low-permeability cuticle covering the animal, fixation of C. elegans tissue for immunoelectron microscopy has proved very difficult. Here we applied a microwave fixation protocol to improve penetr ation of fixatives before postembedding immunogold labeling. Using this tec hnique, we were able to successfully localize several components of yolk (Y P170) trafficking in both wild-type and transgenic strains expressing a vit ellogenin::green fluorescent protein fusion (YP170::GFP). Green fluorescent protein (GFP) and its variants are commonly used as markers to localize pr oteins in transgenic C. elegans using fluorescence microscopy. We have deve loped a robust method to localize GFP at the EM level. This procedure is ap plicable to the characterization of transgenic strains in which GFP is used to mark particular proteins or cell types and will undoubtedly be very use ful for high-resolution analysis of marked structures.