RE: Paraffin embedding and immunocytoche
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From: | "Karen D. Larison" <LARISONK@UONEURO.uoregon.edu> |
To: | histonet@pathology.swmed.edu |
Reply-To: | |
Date: | Fri, 6 Aug 1999 14:24:49 -0800 |
Content-Type: | |
Well, I have had the pleasure of sitting through a couple of graduate-level courses
in physical biochemistry, I think the explanation may be rather simple. We are
pretty watery creatures, and most proteins are adapted to being in an aqueous
environment. Water molecules have a high dipole moment and in constantly bashing
into the protein would prefer to bash into the charged or polar residues. During
normal protein folding, the lipophilic amino acid residues tend to get passively
pushed to the middle of the protein by this constant bashing of the highly ploar
water molecules. When you put the protein into a lipophilic environment such as
paraffin, the charged and polar amino acid residues on the surface need to satisfy
their needs for the opposite charge, which they now can't do by facing outward. In
an aqueous milieu, the dipolar water molecules readily satisfies this need for
charge complementation. Once the water is removed, the protein may need to "turn
inside out" so charge-charge requirements are met. Of course, there may be some
constraints due to the fixation bonds, but the forces that dictate that
charge-charge interactions be fulfilled are pretty darn strong. So my bets are the
proteins "re-conform" to meet this need, and the antigenic epitopes may become
buried. In order to "refold" the protein, and recover the epitope, one needs
water, salts, the proper pH and heat to get the darn protein out of the energy
trough it fell into during the paraffin processing.
Karen in Oregon
Date: Thu, 5 Aug 1999 17:31:54 -0500
From: mark.lewis@shandon.com
Subject: RE: Paraffin embedding and immunocytoche
To: john.e.love1@jsc.nasa.gov, Allison@cardiff.ac.uk
Cc: histonet@pathology.swmed.edu
I've thought about this and have come to the conclusion that heated molten paraffin
is more of a "Dry" heat where the presence of water is virtually absent. Using an
aqueous solution to heat up thi
gs for antigen retrieval is done in a "wet" heat where the presence of water is
abundant... Therefore if we use dry heat we are removing more of the "bound" water
and causing more antigen "masking"
if you like.. Using the wet heat retrieval systems, we do not remove any more of
the molecularly bound water, but possibly add some to the protein systems thereby
"retrieving" the antigenicity if th
antigen...
Just a thought on my part.
Mark Lewis
Technical Specialist
Shandon
1-800-245-6212 ext. 4013
----------
From: Allison@cardiff.ac.uk [SMTP:MIME :Allison@cardiff.ac.uk]
Sent: Thursday, August 05, 1999 3:43 AM
To: john.e.love1@jsc.nasa.gov
Cc: histonet@pathology.swmed.edu
Subject: Re: Paraffin embedding and immunocytochemistry
<<File: ENVELOPE.TXT>>
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John,
Simple question you raised. We are working on the answer - not easy but
very interesting!
If the heat of molten paraffin "destroys" immuno-reactivity, how come we
use heat for antigen retrieval?
Russ Allison, Wales
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