I found that when I applied the primary, then the acid step, then started
from the secondary antibody step, I still had very positive and specific
staining.  I strongly agree with Paul Klosen in that some antibodies are
more difficult to elute that others and that some precipitating chromagens
tend to mask other antigenic sites.

Wendy Rosebury
Cardiovascular Therapeutics
Parke Davis


-----Original Message-----
From: Joyce Kotzuk [mailto:JKotzuk@salud.unm.edu] 
Sent: Monday, April 24, 2000 4:52 PM
To: jkiernan@julian.uwo.ca; histonet@pathology.swmed.edu
Subject: Re: Elution of antibodies??


Would it have to be a different enzymatic label for the second primary
antibody, and if so, why? If acid washing undoes the antigen-antibody
complex, does it take the secondary and link molecules off as well, leaving
just the precipitated DAB or whatever? 
Thanks in advance, 
Joyce Kotzuk, UNM pathology dept.
>>> "J. A. Kiernan" <jkiernan@julian.uwo.ca> 04/23/00 12:09AM >>>
On Thu, 20 Apr 2000, Tim Morken wrote:

> Does anyone have a procedure for the elution of antibodies from tissue 
> sections? Seems I've seen it before on Histonet but I can't find it.

  The answer is acid. Washing at pH below about 3.5 undoes all the
  antigen-antibody complexes but doesn't remove insoluble products
  of enzyme labels such as DAB brown from peroxidase or azo dyes
  from alkaline phosphatase. Having washed out the first lot of
  antibodies, you can then do a second immunostain, with a different
  enzymatic label.

  Sorry I can't provide more details or a few references, despite the
  fact that many exist. My refs database just isn't that well indexed,
  and I don't have an immunohistochemistry book to hand just now.

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1