Re: thanks but i've tried all that

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From:Rob Geske <>
To:Marcia Bentz <>,
Date:Fri, 30 Jul 1999 09:00:17 -0500
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<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>
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