Re: Fixation time for IHC?

From:Todd Sherman

Hello Steve,

Quick questions: Is the tissue from the necropsy unit sitting in 
formalin or other fixative for the entire period prior to your 
acquisition?  Or is it frozen/refrigerated waiting for fixation (heaven 
forbid) for some period and then fixed?  Or is it perfused with fixative 
and then stored temporarily?

If the tissue blocks are large, 24-48 hours in formalin may not be too 
long.  The fixative must penetrate the outer cells and infiltrate them 
before it effects the interior cells of the block.  Is the tissue that 
you are studying "peripheral" tissue or "central" tissue in relation to 
pre-trimmed block shape?  Are the blocks' dimensions measured in cm or 
mm prior to trimming?  It is also possible that if the blocks are large, 
the interior portions may be underfixed.  This may result in 
overfixation for the periphery and inadequate fixation for the center. 
For some proteins that delayed fixation could minimize IHC effectiveness 
as cytolysis proceeds.

FYI:Cross-linking is not really a "problem" but the process of fixation.

HCHO  = pure, monomeric formaldehyde
-CH2- = methylene bridge

A.	protein-H + HCHO ---> protein-HCHOH

B.	protein-HCHOH + H-protein ---> protein-CH2-protein + H2O

(see [John A. Kiernan, 2000]

Also, fixed is fixed.  Once the free amino-acid amine groups have bound 
to fixative solution aldehyde groups and methylene cross-links are 
created to saturation, no more fixation can occur.  This can take some time.

I guess the upshot of all this is that antigen retrieval may not be the 
problem at all.  Could you provide more specifics on the tissue 
acquisition method in more detail?  It sounds as if your antigen 
retrival methods are fine and that fixation may have created some 
problems that you cannot overcome.  What specifically can you not detect 
and in what tissue type?  Someone may be able to provide specifics on 
your particular antigen of interest.


Todd Sherman
HistoSoft Corporation
"Biology in a new form..."
Member Services:

Fixation time for IHC?
From:	"Allen, Steven"

Good morning,
I am doing some IHC on rat tissues (multiple antibodies-one at a time). 
  Due to poor results with several antibodies, I have been speaking with 
technical support and they have recommended a formalin fixation time of 
anywhere between 2 and 12 hours for tissues intended for future IHC.

Our necropsy unit usually waits a minimum of 24 to 48 hours before 
trimming and embedding. I cannot go back and undo the fixation time for 
my tissues, I have to use them. I realize the problem of cross-linking 
with extended formalin fixation. I am performing antigen retrieval: 
0.01M citrate (pH 6.0), 10 min. microwave boil. I am also performing a 
0.16% trypsin digest (30 min) before the blocking step. I have obtained 
positive results with other antibodies, using the same tissues. Also, my 
tissue blocks are about 4 years old.

Could/should I extend the boil time for antigen retrieval?  What kind of 
fixation times do you use for IHC tissues?
Thanks in advance,

Steve Allen
Research Technologist
Lovelace Respiratory Research Institute
Albuquerque, NM

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