Inespecific binding smonard at
Fri Aug 14 19:55:48 EST 1998

Hi Roxana

There is a simple solution to your problem. Incubate with 10% mouse serum
before adding your directly conjugated antibody. If you think about it
after adding your goat anti mouse antibody some of the mouse recognizing
arms of that antibody will be unoccupied. These will readily bind your CD25
as it is also a mouse immunoglobulin hence massive non specific binding.
You could use a CD25 antibody made in another species not too similar to a
mouse (ie not rat unless your goat anti-mouse is absorbed against rat)

Your best bet is too get some mouse serum, inactivate at 56 degrees for 30
minutes aliquot and freeze. When you do your staining do your first two
steps as usual, wash, aspirate supernatent then add about 20ul 10% mouse
serum in PBS, leave at room temp for about 15 minutes then  without washing
add your CD25 antibody.

Good luck

Simon Monard
New York

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