<html><div>Marcia,</div>
<br>
<div>we have had similar background staining with rat kidney. most
of our spurious staining was in the distal convoluted tubules (what is
the distribution of yoru staining?). as i expect you are aware,
kidney is one of those tissues that has a high biotin content. we found
that biotin blocking reduced the amount of background but did not
completely remove it. did the biotin block reduce the background staining
at all? we are currently in the process of doing a second and maybe
a third round of blocking with the assumption that the avidin
concentration in blocking solution is rate limiting. for your
present maintenance of sanity i would suggest (if available):</div>
<br>
<div>1. a primary antibody directly conjugated to a reporter
enzyme</div>
<div>2. a secondary antibody with the reporter enzyme
attached</div>
<br>
<br>
<div>rob</div>
<br>
<br>
<div>At 07:25 AM 7/30/99 -0400, Marcia Bentz wrote:</div>
<div>>Hi all,</div>
<div>>Thanks for the suggestions. Problem is I've already done
everything</div>
<div>>everyone has suggested. At the moment I don't have access to any
other</div>
<div>>tissues. All the products are relatively new (exp. 3/200) but
in</div>
<div>>systematically eliminating things INCLUDING both primary and
secondary</div>
<div>>antibodies, the tissue is still staining. I have blocked for
peroxidase,</div>
<div>>which through testing I found wasn't necessary. </div>
<div>>Here's what I've done:</div>
<div>>substrate alone= no staining</div>
<div>>blocking serum, ABC, substrate= inappropriate staining</div>
<div>>blocking serum,avidin/biotin block (both 15 min and 30 min)=
inappropriate</div>
<div>>staining</div>
<div>>blocking serum, avidin/biotin block, secondary with nml mouse
serum in</div>
<div>>addition to rabbit serum, ABC, substrate= inappropriate
staining</div>
<div>></div>
<div>>I'm using frozen sections (mouse kidney) fixed 10 min with
4%</div>
<div>>paraformaldehyde.</div>
<div>>Pharmingen's rat anti-mouse TNF alpha, Vector avidin/biotin
block, ABC</div>
<div>>Elite rabbit anti-rat IgG secondary, and DAB</div>
<div>></div>
<div>>I want to again stress I'm gettting staining when omitting both
primary and</div>
<div>>secondary antibodies. I'm clueless....</div>
<div>></div>
<div>>Any more suggestions out there?</div>
>
<BR>
<font color="#0000FF"><i><div align="center">
Robert S. Geske<br>
Research Associate<br>
Center for Comparative Medicine and Department of Pediatrics<br>
Baylor College of Medicine</font></i></html>