Folks, In a effort to quench peritoneal macrophage autofluorescence, I resorted to OsO4 fixation and ended up with a mixed bag. I realize that there alternative approaches to the problem of high autofluorescence, but this is the approach I have chosen for at the moment. Procedure Murine peritoneal cells were harvested, washed and incubated with B220-FITC and CD3e-PE, washed again and then either (A) incubated directly in OsO4 (0%, 0.1%, 0.2%, 0.5%, 1%, 2%) in PBS, or (B) first fixed in 1% formalin-PBS (60' in ice, 1 wash with PBS) and then incubated with OsO4. All samples were diluted 1/4, centrifuged and washed 1x with PBS. Results At 0.1% OsO4 in PBS, the autoflu is almost as low as for viable unfixed lymphocytes. However, most of my CD3 and B220 signal intensities are also lost. Several washes did not yield a return of my FITC and PE signals. I got the same results with and without pre-fixation. Questions Not being familiar with OsO4, I'd like to hear from those of you who are. ? Did I quench my fluorochromes (irreversibly?), did my Ab's "fall off", or was my Ag-Ab complex stripped from the surface? ? Would I stand a chance of better results if I were to incubate with the Ab's after the fixation? ? In case of Ab/Ag-Ab losses, would glutaraldehyde fixation be a more "powerful" fix? (I am aware of > autoflu by glutaraldehyde) I'll summarize the responses and their effect on my experiments. Thanks, Jan. GlaxoSmithKline Cytometry Resource 709 Swedeland Road King of Prussia, PA19604 (610)-270-4183
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