RE: Tissue shrinkage
|From:||"Monson, Frederick C." |
References are WAY DOWN, if you want to 'skip to the chase'.
The earliest references I have read on the subject of shrinkage
relate to the effects of dehydration, specifically the transition through
70% EtOH, and heat. But first, let me ask a question.
Your question would lead me to conclude that you believe that you
have a shrinkage problem. Please help us to understand, if that is true,
how you know?
As a suffix to my question, I should say that I believe that Geoff
is correct, and the literature of the '50's through the 'early '70's is
replete with discussions/studies of shrinkage artifact caused by any number
of processing parameters (usually fixation, dehydration, heat and specific
chemistry). I believe that Baker is the source of my original information
about 70% and heat, but many others have also discussed the same subject.
As an example, one of the first things one learns in General Biology
is about 'plasmolysis' in plant "cells". If I were 'obsessed' with the
prevention of shrinkage, I would titrate all of my methods(embedments)
through a group of 'soft' botanical specimens to find those combinations
which, on observation with the TEM, demonstrated the edge between no
shrinkage and plasmolysis - ever-so-slight. Then I would feel more
confident about using the same method in a more shrinkage-susceptible system
in animal cells/tissues.
Methods that do not cause shrinkage in monolayer cultures may do so
in solid culture systems. In the end, we are all 'stuck' with the sure
knowledge that what we 'see' in the electron microscope image requires
interpretation. One could spend a lifetime trying to find the precise
parameters to reduce shrinkage of plasma in blood vessels to near zero.
The consensus appears to be that if shrinkage within a cell or
tissue or block is not minimized, it must, at least, be uniform, and even
that 'feels' too liberal to most of us. The other consensus appears to be
derived from familiarity with our methods. You will note on this list that
there are many questions about problems with standard methods, such as
precipitates that appear in standard mixes. I have yet to see a question
from an investigator who is defining a new method who requires help in
understanding the chemistry of his system. With standard methods there is
experience and inexperience. The list is a teacher with tremendous
If the answers thus far are not really satisfactory, then you must
be more specific about the cellular system with which you are working. Each
one has quirks that only experience can overcome. My experience runs from
end to end of the chordates, but I have to use my references to determine
that for Nematodes (Yuck!!), hot (close to boiling!) fixative/alcohol is
about the only thing that relaxes them so they have a long axis in one plane
for subsequent processing. EM doesn't appear reasonable after such a
beginning, but it has been done. It is even more discomfiting to try to
explain the place of even that kind of consistency in evaluating histologic
(much less the fine) structure in a special group.
Stoward, P.J.(1973), Fixation in Histochemistry, Chapman and
Hall, London, SBN 412-12050 X.
Hayat, M(1981), Fixation for Electron Microscopy, AP, NY,
NY, ISBN 0-12-333920-0.
Frederick C. Monson, PhD
The best research
Center for Advanced Scientific Imaging
occurs before work
West Chester University
at the bench.
West Chester, Pennsylvania, USA, 19383
> From: Geoff McAuliffe
> Sent: Monday, February 25, 2002 5:19 PM
> To: TOMANDBOB@aol.com
> Cc: DebbieSi@bhcs.com; email@example.com;
> Subject: Re: Tissue shrinkage
> TOMANDBOB@aol.com wrote:
> > Histonetters: Does anyone have any ideas/articles/ references on how to
> prevent or minimize tissue shrinkage for specimens going into a
> non-aqueous plastic polymer such as Epon or Spurr? Or is this the
> impossible dream?
> > Thanks,
> > Tom Kuwahara
> > Senior Immunohistochemist
> > Resolution Sciences Corporation
> I have always been told/thought that shrinkage of tissues in epoxy resins
> is much less than in paraffin sections. Proper fixation and subsequent
> processing are your best "defense".
> Geoff McAuliffe, Ph.D.
> Neuroscience and Cell Biology
> Robert Wood Johnson Medical School
> 675 Hoes Lane, Piscataway, NJ 08854
> voice: (732)-235-4583; fax: -4029
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