Richard Haugland richard.haugland at
Fri Feb 15 21:19:20 EST 2002

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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