Re: Clearing agents & immunohistochemistry

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From:"J. A. Kiernan" <>
To:Ian Montgomery <>
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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