Date sent:      	Wed, 03 May 2000 18:21:57 -0400 (EDT)
From:           	BonnieHylander@aol.com
Subject:        	controls for rabbit polyclonals
To:             	Histonet@pathology.swmed.edu

> Plea for help,
>      I am unable to get clean control slides for any rabbit polyclonal 
> antibodies on either paraffin or frozen sections.  There is always background 
> (often very dark) on the collagen, smooth muscle (especially prostate) etc.  
> This happens with commercial antibodies (B2microglobulin or vonWillebrand), 
> in house Abs, as well as with rabbit IgG.  I use casein or superblock. 
> Avidin-Biotin block and exhaust endogenous peroxidase.  I use the ABC kit 
> with DAB.  There is no problem if I omit the primary antibody.  Can anybody 
> offer any insights?  Thank you, Bonnie
>
Bonnie,
 "non specific" binding of polyclonal antisera to connective tissues, 
particularly collagen is not uncommon but it should be possible to 
reduce if not eliminate this by,
1) optimising any antigen retrieval (under digesting or over 
microwaving may be the problem)
2) diluting out primary and secondary antisera to the point where 
only the appropriate epitope will bind antisera. I have found that 
diluting beyond the recommended concentration and incubating  in 
primary overnight at 4oC may give a "cleaner" result. 
3) keep the ABC and DAB as dilute as possible. Using a weak 
solution of DAB is more controlable, just incubate for longer and 
keep an eye on the control.
4) as a last resort, stain in haematoxylin for a little longer or even 
use a "hint" of eosin to mask the offending background.

Any one or all of the above should eliminate this problem.