Hi, I don't think it is the paraformaldehyde as I fix my ACCCHHH!!! biofilms that have GFP with 4% paraformaldehyde and they are very bright for confocal work. BUT I have found that after shooting them using both Argon and Krypton lasers together, the GFP becomes red as well as green, very very bright.... Anyone have any good ideas about why (Howard, are you listening?). Thanks, Deb Berglund Montana State University "Gerstein, Rachel" wrote: > 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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