Hi It could be the fixation step - if the cytokines dont all leak out, why would all the GFP? look into the archives of this list re fixation protocols for GFP - the protein wont retain fluorescence if it gets treated with certain fixatives. good luck, Rachel ======================================================= Rachel M. Gerstein, Ph.D. Department of Molecular Genetics and Microbiology Graduate Program in Immunology/Virology University of Massachusetts Medical School 55 Lake Avenue North Worcester, MA 01655-0002 (508) 856-1044 (508) 856-5920 (FAX) > ---------- > From: James Marvin > Sent: Tuesday, April 9, 2002 2:27 PM > To: Cytometry Mailing List > Subject: GFP along with IL-2 intracellular staining > > > Howdy, > I may have missed a previous discussion on the subject of > intracellular > staining along with detection of GFP. I have looked through the archives > but wasn't able to find what I was looking for. We have a client who is > trying to detect IL-2 intracellularlly along with detection of GFP. They > first fix with 3% paraformaldahyde, then permeabilize with .4% > saponin. When they analyze the sample the GFP is gone. I imagine it is > leaking out of the cell after the permeabilization step. Does anyone have > any tricks to retain the GFP. Altering the construct of the GFP so > that > it will be a surface protein is not really an > option. > > Thanx > > in > > advance > > James > > Marvin > >
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