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From:Gayle Callis <>
Date:Fri, 30 Jul 1999 09:37:45 -0600

Everytime I have used rabbit serum in a blocking reagent, I get background.

you may want to try another species serum, or use BSA in blocking serum with 
the mouse serum instead of the sticky wicket rabbit.  

I have recently dealt with nonspecific background, and trying Jacksons
BSA that is protease and immunoglobulin free, at 1%, can be combined
with goat serum.  I also prefer goat antirat or donkey antirat, with goat
as the first choice for a secondary.  and clean up my normal goat serum
by heat inactivation then microfiltering with 0.45 micron filter.  Do this with
all serums, including mouse.  

A bit of detergent in the buffers, 0.05% Tween 20 may also help and
if it is an fc receptor problem, using F(ab')2 fragment secondaries,
goat antirat, make sure all secondaries are absorbed to mouse, may also
help.  Barb Wright has a very nice endogenous fc receptor buffer that 
is used after all blocks to strip fc receptors from tissues not tried it
but certainly sounded inviting.Secondaries from CalTag, Biosource, 
Jackson Immunoresearch (excellent!) all good, the F(ab')2 fragments.  

I know of one person staining cytokines, who uses mouse antirat F(ab')
IgG from Jackson as a secondary and says he never gets background.  I
presume, with your staining for this cytokine, you are not using saponin.
He did not, used TBS with 0.05% Tween 20 throughout for buffer wash and

Gayle Callis

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