Re: staining plastic-embedded sections

From:Roger Moretz <>

John, Andrea:

I can't speak to all of the stains mentioned, but I
can say that H&E, Masson's, PAS and methyl
green-pyronin all work on JB4/gma sections at 1 to 5
microns.  There are some caveats.  First, the staining
is much lighter for thinner sections (go fig :-));
second--some of the stains are taken up by the
plastic, so there is a tendency for increased
background staining (methyl green/pyronin is the
worst).  The procedure I have used is to not take the
tissue clear to 100% EtOH, but to go into the JB4 from
90%, then pure JB4; polymerize as specified.  Sections
are picked up from a dry glass knife, spread/flattened
on a distilled water bath and picked up on slides. 
Staining is as specified for paraffin sections except
that xylene/clearant is not necessary, but I then go
into water using the alcohol/water routine for
paraffin sections.  Timing for staining has to be
determined by the user, then dehydrate, into xylene
and coverslip.  Methacrylate has the advantage that
alto' there is cross-linking, the matrix is fairly
open, thus available for most of the dyes used in
routine histology, unlike epoxies or resins that are
dense matrices, as well as hydrophobic.  I did most of
this work over 25 years ago when GMA and JB4 were just
being accepted for routine histology.  I haven't had
an opportunity to use these procedures for years now,
but when I did do so, the results were very
satisfactory.  There was quite an extensive literature
published in Stain Technology and J Histochem Cytochem
in the 70's and 80's (plus I think there may have been
1 or more in Lab Investigation, same period).  Check
out PubMed or Index Medicus or one of those for
further references.  Hope this helps.

Roger Moretz
Dept of Toxicology
Boehringer Ingelheim Pharmaceuticals, Inc.
--- "J. A. Kiernan" <> wrote:
> On Thu, 5 Oct 2000, Andrea Voogt wrote:
> > I'm a college student doing a research project on
> the histology of the 
> > garter snake digestive tract and associated
> glands. I already have
> > tissues embedded in plastic, but all the books I
> can find have staining 
> > procedures for paraffin.  Does anyone know the
> staining procedures for
> > plastic-embedded tissues?  The stains I'm hoping
> to use include
> > Mallory-Heidenhain's azan, Best's carmine,
> Gomori's chrome alum
> > hematoxylin-phloxine, Iron-hematoxylin and
> thiazine red staining,
> > Bielschowsky-Foot's method, and Masson's stain,
> but methods for any
> > stains that would be good for digestive tract and
> associated organs
> > would be much appreciated!!!
>    This is a pretty tall order for a beginner! Some
> of those stains
>    can be difficult to get right on paraffin
> sections (for which they
>    are all intended), and you cannot simply apply
> the same procedure
>    to plastic. You mention that the plastic is JB-4.
> This is glycol
>    methacrylate (I think) and it has a chemically
> cross-linked molecular
>    structure, so it cannot be dissolved out of the
> plastic. The
>    embedding medium interferes with penetration of
> dyes and other
>    reagents, and also with the dye-tissue
> specificities.
>    Fortunately It is possible to cut plastic
> sections thinner than
>    paraffin ones, and with a really thin section
> (say, 1 to 2 micrometres)
>    you can see plenty of structural detail with just
> one dye that 
>    stains everything. An alkaline solution of
> toluidine blue is
>    often used. 
>    There are published variants of older staining
> methods that you
>    can use with glycol methacrylate embedding, and
> I'm sure you'll
>    get plenty of good suggestions from the
> listserver, but I'd
>    advise trying something very simple first, and
> not trying to
>    bite off what might turn out to be too much to
> chew. Good luck.
>  John A. Kiernan,
>  Department of Anatomy & Cell Biology,
>  The University of Western Ontario,
>  LONDON,  Canada  N6A 5C1

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