Re: Clearing agents & immunohistochemistry
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From: | "J. A. Kiernan" <jkiernan@julian.uwo.ca> |
To: | Ian Montgomery <ian.montgomery@bio.gla.ac.uk> |
Reply-To: | |
Date: | Fri, 9 Jul 1999 12:01:03 -0400 (EDT) |
Content-Type: | TEXT/PLAIN; charset=US-ASCII |
This is quite a long email, about what clearing agents
may possibly (but hopefully do not) do.
On Wed, 7 Jul 1999, Ian Montgomery wrote:
> Clearing agents for tissue processing in immunohistochemistry.
> Xylene is ok but what about the others, chloroform, benzene,
> toluene, amyl acetate etc. I'm a wee bit suspicious about loss of
> antigenicity with chloroform but nothing I'd like to stake the
> contents of my wifes wallet on.
This is a disturbing question. Let's hope the answer is that
they are all the same. But have there been any published
comparisons? There may well be all sorts of anecdotal
tales of immunohistochemical failures/succeses with pairs of
clearing agents and individual antibodies.
The whole business of diferent clearing agents seems to be
based on individual experiences of small numbers of people
who pass the lore down to the next generation. Much of this
is in the oral tradition, but some of the wisdom finds its
way into books. Those by Culling, Gabe and Lillie are a few
especially notable ones in the mid-20th century literature.
Until today, the desirable properties of a clearing agent
related to things like transparency, hardening and shrinkage
of the specimen, tolerance of water in the preceding alcohol,
and effects on the cutting properties after paraffin
embedding. Even the density was important: objects cleared
in chloroform don't sink. The more viscous liquids, with
cedarwood oil as an extreme example, are not completely
removed from the specimen during wax infiltration, and this
(traditionally and anecdotally) allows the cutting of
sections that really are less than 4um thick. (A microtome's
setting should always be questioned, especially if it isn't
putting out perfect ribbons without any cracks or other
marks in the tissue.)
With Ian's perspicacious question we now have to ask if
clearing agents affect the chemistry as well as the physics
of tissue processing. Can a clearing agent modify an epitope
in such a way as to make it unrecognizable after rehydration?
Clearing agents that are trade secrets, not disclosed even in
patents, cannot be discussed in this context. Such liquids
should never be used. The known compounds used for clearing are
extremely unlikely to induce chemical changes in proteins or
carbohydrates, but they might change the conformation of
fixed and dehgydrated macromolecules in some hitherto
unsuspected way. Ian, you have made a suggestion that could
be developed into a useful research project. It's a pity that
these days most of the money is given to people who never see
the cells they work with.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
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