Re: Paraffin temp for immuno's

From:Ross Stapf

On searching the archives I found a thread which discusses possible reasons why heat in paraffin during processing might be more detrimental than heat in antigen retrieval solution.  

But, I can't find if anyone came to a conclusion that this was true and any damage was being done.  Also if any damage is done, do our antigen retrieval methods reverse/undo/unmask this damage.

I'm sure someone has studied this in order to come up with this 56

Ross Stapf

Karin K. Larison wrote in August of 1999:
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
Subject:       RE: Paraffin embedding and immunocytoche
To:  ,

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

Just a thought on my part.

Mark Lewis
Technical Specialist
1-800-245-6212 ext. 4013

Sent:  Thursday, August 05, 1999 3:43 AM
Subject:  Re: Paraffin embedding and immunocytochemistry

 --------------------------------------------------------------------------  --
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

<< Previous Message | Next Message >>