Ken may well be right that there could be some problems that I had not considered in proposing the anti-fluorescein system in this case. There could/would be some residual binding of a fluorescein annexin V to the anti-fluorescein in the second step. Although anti-fluorescein quenches over 95% of the fluorescence of most fluorescein conjugates, this could still result in some false signal in non-apoptotic cells. We have prepared a green-fluorescent Alexa Fluor 488 annexin V that has no crossreactivity with anti-fluorescein that should eliminate that problem in this case or some other cases. The overall principle is to shift an available fluorecein conjugate's fluorescence to longer wavelength so that a different green-fluorescent dye that is available could be used for for the green-fluorescent signal. Using the non-crossreacting Alexa Fluor 488 dye in the detection scheme with a red-fluorescent anti-fluorescein should then work, with staining done in either order. Kenneth Ault wrote: > I would be concerned, in the technique described by Richard Haugland, > that there would be free anti-fluorescein binding sites available to > incorrectly bind the second fluorescein conjugated antibody. I have > never used anti-fluorescein in this type of protocol, but I know from > bitter experience that anti-immunoglobulins cannot be used in this way > unless those free binding sites are blocked using excess > immunoglobulin. I'm not saying it won't or can't work, just that one > should be careful to confirm the specificity of the second reagent in > such a protocol. Ken Ault
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