Re: Clearing agents & immunohistochemistry

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From:"Barry Rittman" <>
To:histonet <>
Date:Fri, 09 Jul 1999 13:49:09 -0500
Content-Type:text/plain; charset=us-ascii

            I think your comments are on the mark. When we tried to
compare lectin binding (several lectins) on paraffin section from our
lab with another lab we found there were major differences in  binding.
We used chloroform and they used xylene in their processing to paraffin
wax, everything else was the same.
As you noted, there may be conformational changes. Also many people
forget that many of the carbohydrates are present as glycolipids and
glycoproteins and when lipid solvents are used there may be extraction
of some of  these and/or conformational changes.

"J. A. Kiernan" wrote:

>      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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