Re: Paraffin temp for immuno's

From:Patsy Ruegg

As usual John enlightens us all.  With the advent of HIER methods to
unmask proteins cross-linked by formalin our understanding of the tissue
biochemistry has changed, if we were not crosslinking the proteins with
formalin to begin with, we would not have to go through all the tissue
altering procedures (impossible to standardize from lab to lab) we do to
access the antigen site.  I have for years advocated avoiding !0%NBF for
IHC studies.  I have used zinc formalin myself for years and although it
is not formaldehyde free, it does not require near the retrieval 10%NBF
does.  Enough for this soap box, I am afraid we have lost the battle to
change the 10% NBF choice of a standard fixative.  We prefer too much to
look at archieval material which is already prepared this way, so we have
to jump through hoops trying to get at those antigens and reproduce IHC
results, hopefully in a standard way so the results can be compared from
shop to shop.  Nobody ever said this was easy, did they???

"J. A. Kiernan" wrote:

> Ross Stapf (or was it Patsy Ruegg) wrote:
> > On searching ... why heat in paraffin ... might be more
> > detrimental than heat in antigen retrieval solution.
> > ... can't find if anyone came to a conclusion that this was true ...
> > I'm sure someone has studied this in order to come up with this 56
> The cited temperature 56C must mean that this is another urban
> laboratory
> legend!  Anyone studying effects of wax temperature would
> investigate
> steps such as 50-55-60. An investigation with a precision of one
> degree would need 20 lots of trials to cover the range, and what
> agency would fund such a study?
> Several anecdotal Histonet replies have reported exactly the
> opposite - that temperatures well above the mid-50s does more
> good than harm when it comes to detecting antigens
> immunohistochemically.
> These informal reports, based on firt-hand experience, carry much
> more
> weight than "someone said that..." or a photocopy of some piece
> of
> paper found in a cardboard box.
> There is also a Common Wisdom of Immunohistochemistry that says
> coagulation of proteins by heat or coagulant fixatives liberates
> the epitopes of insolubilized protein molecules.
> If the routine fixative everywhere was a simple alcohol-acetic
> mixture, would there be any of these problems with masked
> antigens?
> --
> -------------------------
> John A. Kiernan
> Department of Anatomy and Cell Biology
> The University of Western Ontario
> London,   Canada   N6A 5C1

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