We have had similar problems, using a soluble YFP protein. However, we found that the attached protocol works quite well. It depends on fixation and permeabilization followed by secondary detection of the GFP/YFP using polyclonal Abs. It seems that the fix does help retain the GFP, but destroys its endogenous fluorescence. But, at least in our case, it retained sufficient immunoreactivity to be easily detected with a a polyclonal Ab. If anyone is interested, write me and I can send you some sample data. I hope this helps. By the way, this insight and solution was developed by Anja Hauser in my group. Mark Shlomchik At 3:24 PM +0800 5/5/06, Dr. Tomas Corcoran wrote: >Hope someone can help with this. > >We are looking at Coll-1 and SMA in GFP positive cells. >Unfortunately, when we permeabilise to lable the SMA / Coll-1 we get >significant leakage of our GFP and lose our ability to gate the >cells. Has anyone any ideas or experience to help us resolve this? > >Much Appreciated > >T > >-- >No virus found in this outgoing message. >Checked by AVG Free Edition. >Version: 7.1.392 / Virus Database: 268.5.3/331 - Release Date: 03/05/2006 -- Mark Shlomchik, MD, PhD Professor of Laboratory Medicine and Immunobiology Yale University School of Medicine 203-737-2089 203-785-5415 (fax) mark.shlomchik@yale.edu OFFICE ADDRESS FOR FEDEX Yale University School of Medicine 1 Gilbert St. TAC S541 New Haven, CT 06510 ADDRESS FOR POSTAL LETTERS (allow two weeks for delivery) Yale University School of Medicine 333 Cedar St. Box 208035 New Haven, CT 06520-8035
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