Marcia,

Have you tried different blocking agents, such as Dako's non-protien 
blocking agent or Zymeds CAS blocking system? Even powdered skim milk 
solutions work for blocking (casien).

You had said you had good staining with this method on similar tissue before 
so you may have to just accept your tissue has been ruined somehow. I know 
it's tough to tell a researcher that but it happens occasionally.

Tim Morken, B.A., EMT(MSA), HTL(ASCP)
Infectious Disease Pathology
Centers for Disease Control
MS-G32
1600 Clifton Rd.
Atlanta, GA 30333
USA

email: tim9@cdc.gov
       timcdc@hotmail.com

Phone: (404) 639-3964
FAX:  (404)639-3043


----Original Message Follows----
From: Marcia Bentz <mb7x@virginia.edu>
To: histonet@pathology.swmed.edu
Subject: thanks but i've tried all that
Date: Fri, 30 Jul 1999 07:25:55 -0400

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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