RE: [Histonet] fixation/no fixation

From:"Morken, Tim - Labvision"

George asks (again) 
<< The questions were 

1) what do you do to make fixed tissues useable for these studies 

There's two parts to this, one involving the tissue, one the antibody
First, fixed tissues, specifically formalin-fixed tissues, are being
"treated" with various enzyems (trypsin, proteinase K, etc) or heated
buffers at various pH's and temperatures in order to "somehow" "unmask" the
epitopes. Very vague, really, but there is some research being done and
various explanations, from breaking formalin-induced bonds to restoring
electrical charges. I was just at a histochemistry meeting in San Diego
where all these explanations went head-to-head. It was very interesting and
I think the "restoring charges" explanation is edging ahead. 

Second, we are screening antibodies to find the ones that work best on
formalin-fixed tissue although they may still require some "pretreatment."
Indeed, many of these antibodies don't work at all on frozen tissue.

2) what is the comparable results between fixed and non fixed tissues in the
results? >>

Since formalin-fixed tissue shows decent morphology, it is preferred over
frozens if the target can be detected. Many times we really do want to see
the tissue structure where an antibody is found, not just the general
location of an antibody. Since freezing can destroy cell and tissue
boundaries, many protiens (and their target epitopes) are translocated
during the thawing that occurs after the freezing.  So location is not
necessarily exact in frozen tissue. In my experience, tissue that is
properly fixed gives great results - strong signal, exact location and
excellent morphology. And many of the targets once detected by
histochemistry can now be detected by antibodies, thus making frozens even
less necessary.

But the primary moving force for fixed tissue is the clinical pathology need
- if a test can be done on fixed tissue it makes life easier for everyone,
plus you may have a (nearly) permanent paraffin block to use in the future
for other tests.

Tim Morken
Lab Vision - Neomarkers

Free webhosting for US State Histotechnology Societies:

-----Original Message-----
From: George Cole [] 
Sent: Wednesday, August 11, 2004 8:52 AM
Subject: [Histonet] fixation/no fixation

Thank you for your responses to my question.  They reflect a much broader
range of tissues and entities studied than my muscle/nerve/kidney studies.
My questions arose from my  experience over 27 years with  biopsy tissues of
muscles,  nerves and  kidneys. The question simply was, given, that once
fixation was considered impossible for use with immunofluorescent work and,
I thought, all antibody techniques. Now the histonet carries constant
references to fixation in these studies. The questions were 1) what do you
do to make fixed tissues useable for these studies and, 2) what is the
comparable results between fixed and non fixed tissues in the results? I am
impressed with the broad responses reflecting much expertise in work done.
But questions 1 and 2 have been whiffed aside by an acceptance of the fixed
tissue work, and  this retiree is still in the dark about what has altered a
once Absolute Rejection of fixation, and allowed histotechs of today an
acceptance of fixation in certain areas of histochemistry.  Like the food
ladeller and Oliver Twist: When Oliver asks, "Please, sir. May I have some
more?", the man serving the children shouts; "More! The boy wants more!
Surely the boy shall hang!!" That I should  question fixatives seems  worse
than Oliver asking for more
oatmeal!   The question about fixatives seems foreign to  the current
generalized mind set with strong habits of  use of them.  I ask these
questions after a working lifetime in which fixatives caused absolute
failure in all but a few processes in muscle work and it was taken as an
additional absolute, that fixatives ruined imnunofluorescnce in kidney work.
Have these absolutes  evaporated?                
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