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 penetration of fixatives before postembedding immunogold labeling. Using this technique, we were able to successfully localize several components of yolk (YP170) trafficking in both wild-type and transgenic strains expressing a vitellogenin::green fluorescent protein fusion (YP170::GFP). Green fluorescent protein (GFP) and its variants are commonly used as markers to localize proteins in transgenic C. elegans using fluorescence microscopy. We have developed a robust method to localize GFP at the EM level. This procedure is applicable to the characterization of transgenic strains in which GFP is used to mark particular proteins or cell types and will undoubtedly be very useful for high-resolution analysis of marked structures.
Paupard
M C
MC
Center for C. elegans Anatomy, Department of Neuroscience, Albert Einstein College of Medicine, New York, New York 10461, USA.
Miller
A
A
Grant
B
B
Hirsh
D
D
Hall
D H
DH
D000818:Animals; D030801:Animals, Genetically Modified; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000235:genetics*; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008872:Microwaves; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D016707:Tissue Fixation / Q000379:methods; D014819:Vitellogenins / Q000235:genetics / Q000378:metabolism*
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014819:Vitellogenins; D049452:Green Fluorescent Proteins