Journal
JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY
Volume 49, Issue 8, Pages 949-956Publisher
SAGE PUBLICATIONS LTD
DOI: 10.1177/002215540104900803
Keywords
immuno-EM; GFP; microwave fixation; C. elegans; yolk
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Funding
- NCRR NIH HHS [R24 RR012596, RR12596] Funding Source: Medline
- NIGMS NIH HHS [F32 GM019167, F32 GM019167-02, F32 GM019167-01] Funding Source: Medline
- NIH HHS [R24 OD010943] Funding Source: Medline
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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.
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