RE: thanks but i've tried all that

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From:Cynthia Favara <>
To:"" <>, "'Marcia Bentz'" <>
Date:Fri, 30 Jul 1999 09:43:05 -0400

	I have missed most of this conversation, but here are my thoughts.
Maybe the ABC Elite and DAB combination is to concentrated for the tissue. I
think I would try to stain without the avidin-biotin step ie use and HRP
conjugated secondary. This may be sufficient. I have diluted the ABC elite
ie use more than 5 ml of buffer per solutions. Also when I get a reaction
that is way to strong I use AEC which I make up myself and decrease the
amount of H202 so the reaction goes more slowly. 
	If this is rehash please disregard and have a good weekend.

Cynthia Favara
Rocky Mountain Laboratories
903 S 4th Street
Hamilton, MT 59840
ph: 406-363-9317
FAX: 406-363-9286

> ----------
> From: 	Marcia Bentz[]
> Sent: 	Friday, July 30, 1999 5:25 AM
> To:
> Subject: 	thanks but i've tried all that
> 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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