I suggest that maybe you need a light fixation of some sort. It could be
that in the time period between sacrificing, perfusing and freezing that the
epitopes you are after are degraded. 

Tim Morken
CDC, Atlanta

-----Original Message-----
From: Chris van der Loos [mailto:c.m.vanderloos@amc.uva.nl] 
Sent: Wednesday, March 12, 2003 3:08 AM
To: histonet@pathology.swmed.edu
Cc: nmuvarak@facstaff.wisc.edu
Subject: RE: Heat-Induced Epitope/Antigen Retrieval


Dear Nidal,
No, you are absolutely right. Tissue treated as you described shouldn't be 
HIER treated. It will probably destroy it completely.
Please be aware that storage of your sections at -20C may have a 
deleterious effect on some epitopes. This at least happened here after 2-4 
months storage of cryostat sections with CD4 and CD3.
The background staining as you described might be caused by the fact that 
both MMP-2 and -9 are enzymes released to the tissue and therefore doesn't 
need to be nicely localized inside a cell.

Chris van der Loos
Dept. of Cardiovascular Pathology
Academic Medical Center
Amsterdam, The Netherlands

Nidal Muvarak wrote:
 >Date: 11 Mar 2003 17:38:10 -0600
 >From: NIDAL E MUVARAK 
 >Subject: Heat-Induced Epitope/Antigen Retrieval.
 >Hi. I'm doing MMP-2 and MMP-9 IHC on mouse lung tissue. I haven't been
>successful at getting good, consistent staining.... either no staining or 
too
 >much background. A few people suggested HIER, however, the tissue is not
>formalin-fixed, nor paraffin-embedded. Once I harvest the tissue, I perfuse
>the lungs (through the pulmonary arteries) with a 50:50 mixture of OCT
 >(TissueTek) and PBS. Then I freeze them in OCT. Then I section them at 4 
or 5
 >microns in thickness. I store the slides at -20C and when I'm ready to do
an  >experiments, the first thing I do is fix them in cold aceton for 10-15
min,  >dry the slides for 5-10 min, then rehydrate (on a shaker to remove
the 
OCT) in
 >either dH2O or TBS, depending on the stain I'm doing. So, does anyone 
think I
 >still need to do the HIER step? From what I read about HIER is that it's 
used
 >mainly with FFPE tissue, since that's what masks the antigenicity. Thank
you  >for your time.  >Nidal E Muvarak  >Associate Research Specialist
>Department of Biomedical Engineering  >University of Wisconsin-Madison
>1550 Engineering Dr.; Rm. 2158  >Madison, WI 53706-1609
 >Lab: (608) 265-8921; Office: (608) 265-4205;
 >Home: (608) 256-7934; Cell: (608) 332-6068