At 03:57 PM 1/26/99 -0500, you wrote: > >This message brings up a point that I'd like to see addressed by the experts. >In my hands, FITC (as well as PE, PerCP, APC, PI, 7-AAD, and ToPro-3) is very >stable, and no precautions need to be taken while staining or running samples, >as no degradation has ever been seen. Every clinical and research lab I've been >in stains samples on the bench top and takes no special precautions to avoid >any forms of light. But some researchers here do things like staining in the >dark, encasing samples in aluminum foil, and using red filters on the room >lights in the flow lab for fear of fluorochrome degradation. Based on my >experience, I'm not sure why they fear the light to this extent. I've seen this >happen using the fluorescent scope, but I think those are usually fluors like >Cy2 and Cy3. Are some derivations of fluorescein more photolabile than others, >or am I ruining my samples. > >Keith Bahjat >kbahjat@ufl.edu > I don't claim to be an expert on photobleaching, nor have I done any controlled studies on the topic, but here's my impression. Most of us who have taken photomicrographs of immunohistochem slides stained with FITC have probably experienced photobleaching of the green fluorescence; i.e., by the time you have focused and determined what field you want to shoot, the green fluorescence has diminished. In fact, mounting compounds are marketed to counteract photobleaching. However, I do all of my staining for flow work on the benchtop in a normally lit lab, with no apparent photobleaching. We do, however wrap our stained samples in Al foil if they are to be sitting around exposed to light (in a cold room, for instance) for any appreciable length of time. And we run the samples in a flow lab with dimmed lights. This is probably not necessary, but reduces eye strain from reflected glare off of the computer screen and makes my "customers" feel their samples are safe. I think the reason we see photobleaching in immunohistochem slides, but not so much in flow samples, has to do with the relative intensity of the illuminating light, and perhaps the wave length and cumulative time of exposure. Compared to normal room light, the mercury arc lamp of an epifluorescence scope is really blasting away at the FITC molecules.
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