Re: thanks but i've tried all that

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From:Karen S Pawlowski <>
To:Marcia Bentz <>
Date:Fri, 30 Jul 1999 09:20:31 -0500 (CDT)
Content-Type:TEXT/PLAIN; charset=US-ASCII


Since you get labeling with blocker and ABC along, it sounds like your
blocking serum may not be strong enough-'tho I don't
know why.  I do immuno's on really sticky tissue-processed in celloidin-
and I have gone to a 10% normal serum, 10% nonfat dry milk, 1% BSA
solution in BSA to get rid of the stickyness.  Try it, if it works then
your material, for some reason was absorbing the ABC indiscriminately.  If
it doesn't work, then I would suspect that your biotin blocking isn't
strong enough.  Don't bother with the other steps until you've gotten this
part worked out=no staining.

Hope this helps.


On Fri, 30 Jul 1999, Marcia Bentz wrote:

> Hi all,
> Thanks for the suggestions. Problem is I've already done everything
> everyone has suggested. At the moment I don't have access to any other
> tissues. All the products are relatively new (exp. 3/200) but in
> systematically eliminating things INCLUDING both primary and secondary
> antibodies, the tissue is still staining. I have blocked for peroxidase,
> which through testing I found wasn't necessary. 
> Here's what I've done:
> substrate alone= no staining
> blocking serum, ABC, substrate= inappropriate staining
> blocking serum,avidin/biotin block (both 15 min and 30 min)= inappropriate
> staining
> blocking serum, avidin/biotin block, secondary with nml mouse serum in
> addition to rabbit serum, ABC, substrate= inappropriate staining
> I'm using frozen sections (mouse kidney) fixed 10 min with 4%
> paraformaldehyde.
> Pharmingen's rat anti-mouse TNF alpha, Vector avidin/biotin block, ABC
> Elite rabbit anti-rat IgG secondary, and DAB
> I want to again stress I'm gettting staining when omitting both primary and
> secondary antibodies. I'm clueless....
> Any more suggestions out there?

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