Linked Life Data

12502749

Source:http://linkedlifedata.com/resource/pubmed/id/12502749

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2002-12-27
pubmed:abstractText
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.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0022-1554
pubmed:author
pubmed:issnType
Print
pubmed:volume
51
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
5-14
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2003
pubmed:articleTitle
Simultaneous detection of EGFP and cell surface markers by fluorescence microscopy in lymphoid tissues.
pubmed:affiliation
Trudeau Institute, Saranac Lake, New York 12983, USA. trandall@trudeauinstitute.org
pubmed:publicationType
Journal Article, Comparative Study, Research Support, U.S. Gov't, P.H.S.