[Histonet] Universal (protein) block for IHC

[Johnson, Teri]

For antibody staining in clinical labs, antibodies and detection systems
have been well characterized and optimized for use in human samples.
When we automated the immunostaining in the clinical labs I worked in,
we never used it there either.

Performing antibody staining on animal samples, especially polyclonal
antibody staining, tends to bring more background into the picture.
Unfortunately there are times when we have very small amounts of sample
(be it antibody or specimen) to do our initial workups, and therefore
really need to start with a system that gives us the greatest chance for
success with little to no background staining.  When using any
avidin/biotin system, we routinely do a biotin block.  We know not every
tissue sample will have endogenous biotin causing non-specific staining,
but it just keeps us from having to include a control to account for
that variable alone. Same goes with the protein block.

Undoubtedly, many of us continue to do things in protocols "because
we've always done it".  When trying to obtain staining using antibodies
which may have never been tested in IHC against species also never
tested, which we do a lot of,  I tend to lean toward the side of what
can theoretically produce the best possible outcome the first time out
of the gates.

We would need to do some serious side-by-side in-house development for
me to be comfortable dropping it altogether in this environment.

Saw a quote that I dearly love--don't know who to attribute it to:
"I want to live in Theory--everything works there."

Best wishes,

Teri Johnson
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, Missouri  64110
tjj <@t> stowers-institute.org



-----Original Message-----
From: Richard Cartun [mailto:Rcartun <@t> harthosp.org] 
Sent: Thursday, April 07, 2005 1:08 PM
To: histonet <@t> lists.utsouthwestern.edu; Johnson, Teri
Subject: Re: [Histonet] Universal (protein) block for IHC


I find it interesting that some labs still use a protein or normal sera
block.  We stopped doing this years ago (for paraffin sections) and we
never see any nonspecific staining.

Richard

Richard W. Cartun, Ph.D.
Director, Immunopathology & Histology
Assistant Director, Anatomic Pathology
Hartford Hospital
80 Seymour Street
Hartford, CT  06102
(860) 545-1596
(860) 545-0174 Fax

>>> "Johnson, Teri" <TJJ <@t> Stowers-Institute.org> 04/06/05 05:34PM >>>
Has anybody converted to using a universal protein block (casein) for
their immunohistochemistry protocols rather than using species specific
normal sera?  For those who have made the conversion, did you have to
make any changes to your methodology or did you just plug it in to your
protocol and run?  Did you notice any difference in the staining
intensity for known antibodies (stronger, weaker, no change)?  Are you
also using it as a secondary antibody diluent or for washes?  Finally,
are you using commercially available reagent or are you making your own?
 
Thanks so much!
 

Teri Johnson
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, Missouri  64110
tjj <@t> stowers-institute.org 


 
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