BAckground with mouse antibodies

From:Gayle Callis <>

You wrote:  I am having an awful time with these two stains, would some
wise person tell 
me what I am doing wrong?  I am using FFPE spleen as a control.  I seems to 
stain wheither I use primary antibody or not.  I have tried blocking with 
serum-free and also 10% normal serum with 5% BSA in buffer.  I am using
Envision plus mouse kit (HRP).  I block with their peroxidase blocking 
solution.  Any ideas?

Background can be caused by fc receptors.  If your primary is rat antimouse
CD20 or 21, then a good block is 5 - 10% goat with 2.5 - 5% mouse serum, 30
min.   The secondary can be diluted in this normal serum block AND the
secondary should be an F(ab')2 fragment of IgG ie goat antiRat F(ab')2 -
IgG.  Make sure you have the secondary diluted out properly, often a
0.5mg/ml concentration works well at 1:250, but you may have to do a
dilution panel on a secondary.  

We prefer goat antiRat vs rabbit antiRat since the rabbit is a sticky
critter, we have better luck with a goat secondary, but a rabbit antiRat
can be tweaked the same way. 

Also, put Tween 20 in your buffer, 0.05% to start with, can go up to 0.2%
or so. 

Good luck

Gayle Callis
Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
Bozeman MT 59717-3610

406 994-6367
404 994-4303 (FAX)

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