Re: Bone-GFP again

From:Gayle Callis

This group did decalcified bone i.e. mouse nasal turbinates, access
publication for details. 

Simultaneous Detection of EGFP and Cell Surface Markers by Fluorescence
Microscopy in Lymphoid Tissues 
Kim L. Kusser and Troy D. Randall, 
Enhanced GFP (EGFP) is a powerful tool for the visualization of tagged
proteins and transfected cells and is easily detected by fluorescence
microscopy or flow cytometry in living cells. However, soluble EGFP
molecules can be lost if cell integrity is disrupted by freezing,
sectioning, or permeablization. Furthermore, the fluorescence of EGFP is
dependent on its conformation. Therefore, fixation protocols that
immobilize EGFP may also destroy its usefulness as a fluorescent reporter.
Here we determined which methods of preparing murine lymphoid tissues
immobilized soluble EGFP protein and retained its fluorescence while
simultaneously maintaining the antigenicity of various immunologically
important molecules and best
preserving the overall morphology of the tissues. We found that EGFP could
not be visualized in frozen sections of spleen that had not been fixed
before freezing. However, robust EGFP fluorescence could be observed in
frozen sections of tissues fixed under various conditions. Fixation was
important to immobilize EGFP rather than to maintain conformation, because
only minimal EGFP could be detected by immunofluorescence in unfixed frozen
sections. Although it had little effect on EGFP fluorescence, the inclusion
of sucrose during fixation better preserved the morphology of fixed
tissues. These methods also preserved the antigenicity of a wide variety of
molecules used to identify cell types in lymphoid tissues. (J Histochem
Cytochem 51:514, 2003) 

Key Words: EGFP, frozen sections, immunofluorescence, mouse, spleen,
perfusion, cell surface markers 

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)


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