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> I am occasionally noticing a reduction in fluorescence
> intensity with increasing antibody concentration at levels beyond
> saturation.
With indirect staining, when the first antibody is above saturation,
extra rinses may be necessary before applying the second antibody.
Otherwise, the residual unbound first antibody may neutralize the second
antibody in solution before it ever binds to the cells, thereby reducing
the fluorescence intensity. The extent of this artifact would of course
depend on the concentration of second antibody employed. This artifact
would not, however, occur with direct conjugates. I have no explanation
for a reduced intensity at higher concentriations of these.
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Eric Martz, Professor of Immunology emartz@microbio.umass.edu
Dept Microbiology Voice: 413-545-2325 FAX: 413-545-1578
Morrill IVN 203, Box 35720, Univ Massachusetts, Amherst MA 01003-5720
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