I would be very surprised if this fixation approach worked. Proteins can be fixed inside a cell because they can be 1.) cross-linked or 2.) made insoluble by the fixation process; this latter process has been termed coagulative fixation and is due to partial denaturation. A small organic molecule like a drug, is unlikely to be able to be cross linked by PFA and will not denature as a protein would. If you got lucky and by chance did have a drug that had a site that was amenable to cross linking, it would likely disrupt the Ab binding site. Proteins work in part because they are large molecules and you can fiddle with one part without disrupting the Ab binding site. Call me Mr. Optimist! Calman Prussin Laboratory of Allergic Diseases NIAID/ National Institutes of Health ---------- From: Richard K. Meister Sent: Wednesday, May 28, 1997 3:20 PM To: cyto-inbox Subject: Drug studies Hi, everyone: I have an investigator who wants to look at intracellular distribution of a drug using confocal laser scanning microscopy. The drug is not fluorescent, so he has a monoclonal antibody (FITC conjugate) to the drug. Our question is, during permeablization to allow the antibody into the cells, what is the best way to prevent the drug from leaching out of the cells? I suppose a pre-permeablization paraformaldehyde fixation step would be helpful, but I would like to hear from anyone who has done such drug studies and knows if this is an effective approach. Thanks in advance.
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