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Visualization of identified GFP-expressing cells by light and electron microscopy
Katherine Luby-Phelps1, Gang Ning, Joseph Fogerty
1Department of Cell Biology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. kphelps@mcw.edu
Summary
We developed a method to visualize Green Fluorescent Protein (GFP) in fixed tissues using LR White embedding. This technique preserves GFP fluorescence and antigenicity for both light and electron microscopy, enabling precise cellular localization.
Area of Science:
- Cell Biology
- Microscopy Techniques
- Biochemistry
Background:
- Green Fluorescent Protein (GFP) is a vital reporter in molecular biology.
- Visualizing GFP expression at the ultrastructural level is challenging.
- Existing methods often compromise tissue morphology or GFP signal.
Purpose of the Study:
- To establish a robust protocol for visualizing GFP expression in fixed tissues.
- To enable correlative light and electron microscopy of GFP-labeled cells.
- To maintain both GFP fluorescence and antigenicity for multi-modal imaging.
Main Methods:
- Tissue fixation using paraformaldehyde/glutaraldehyde.
- Embedding in LR White resin.
- Direct fluorescence microscopy of 1-micrometer sections.
- Immunogold labeling with anti-GFP antibodies for electron microscopy.
Main Results:
- GFP fluorescence is retained after fixation and LR White embedding.
- Unstained sections allow direct visualization of GFP.
- Immunogold labeling successfully localizes GFP at the ultrastructural level.
- Tissue morphology is preserved for subcellular analysis.
Conclusions:
- The developed protocol effectively visualizes GFP expression in fixed tissues.
- This method facilitates high-resolution localization of GFP using correlative microscopy.
- It offers a valuable tool for studying gene expression and protein localization in cellular contexts.