[Histonet] Rabbit Anti-sera Background

[Richard Bruggeman]

We are having some problems with extensive background and nonspecific
staining with a new rabbit polyclonal anti-sera.  Here are some of the
staining details:
 
-FFPE human tissue
-1mM EDTA pH 8.0 HIER for 20 minutes in steamer seems to provide best
staining
-10% H2O2 block for 10 minutes @ RT (standard for our lab)
-block with 1% BSA in TBS for approx. 30 minutes @ RT (no rinse
afterwards, just drain off blocking reagents)
-primary antibody dilution worked out to 1:1000-2000 (diluted in Dako
diluent) for 30 minutes @ RT via titration runs

-Dako Envision+ Goat Anti-Rabbit/HRP Labeled polymer detection used for
30 minutes @ RT (standard for our lab)
-Dako DAB+ for 10 minutes @ RT (standard for our lab)
-counter stained w/ Dako Hematoxylin for 1-2 minutes (standard for our
lab)
-All rinses done in  Dako TBS w/ 0.05% Dako Tween 20 (standard for our
lab)
 
 
We have tried doing peptide blocking experiments that have shown some
of the staining can be blocked, however most of the staining is still
present, suggesting that it is due to nonspecific binding.  We know from
Westerns that our blocking experiments include a VERY large excess of
peptide, so all of the specific antibodies should be bound to peptide. 
Negative controls (without antibody and/or with peptide only) always
show up DEAD negative.  No avidin/biotin to worry about since this
detections system is a labeled polymer.  The detection system has been
working very well for us in the past, including with other commercially
available rabbit polyclonals, so I can't imagine this would be the cause
of our problem.  What suggestions might you have to reduce this intense
background/nonspecific staining.
 
Richard "Trey" Bruggeman
 
Milton S. Hershey Medical Center
Penn State College of Medicine
Department of Pathology
500 University Drive
Hershey, PA  17033
 
(717) 531-1044