Mouse lung MMP IHC background problems

From:Gayle Callis

Chris van der Loos is correct, you will not need antigen retrieval with
acetone fixed lung tissue.  

One of our research labs works exclusively with mouse lung tissue, prepared
the lung in same way (fill with OCT - they do not dilute the OCT) but we do
store blocks and infixed frozen sections at -80C, often for weeks.  We also
air dry  much longer before freezing down sections and after removal from
freezer prior to fixation. Chris van der Loos had a wonderful discussion on
how he does frozen sections for storage, check Histonet archives  - his
sections are dried longer also. 

There are other sources of background and mouse lung is notorius for this -
our lab uses Alkaline phosphatase IHC, with NBT/BCIP chromogen - very
sensitive.  They maintain HRP IHC methods produces more background than AP
in the mouse lung.  

You did not say WHAT immunostaining method you are using?? Please enlighten
us, the problem may be in your staining method rather than tissue or
section preparation. 

The Glucose oxidase block for endogenous peroxidases/pseudoperoxidases is
superior on minimally fixed (acetone) frozen sections without damaging
morhphology. It quenches all completely. 

Also using secondary antibodies that are F(ab')2 fragments of IgG reduces
nonspecific binding to fc receptors on tissues - blocking is also critical
- there are many sources of background and poor staining.


Nidal Muvarak wrote:
 >Date: 11 Mar 2003 17:38:10 -0600
 >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 
 >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 
 >mainly with FFPE tissue, since that's what masks the antigenicity. Thank you
 >for your time.
Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)


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