Re: staining plastic-embedded sections

From:Kim Kusser <>

I do alot of IHC, but rarely do much paraffin or plastic. However, 
that's not to say I haven't done plastic.

I recently have done several projects in which I embedded
in plastic using the same kit as you. Along with a simple
crystal violet stain,  I made up MacNeal's which is a tetrachrome 
(4 color) stain.

Now, you'll have to time how long you keep the stain on there, and
the subsequent washing steps to determine what give you the best
colors. Usually, I cut several sections (I was doing mouse liver
and spleen...which stain differently from each other), and float them
on drops of water that are on the slide. 

Once I get enough on the
slide, I take the slide over to the slide warmer, and let the water evaporate.
This sticks the section to the slide. I then place the MacNeal stain on the slide,
and leave it for whatever amount of time I think it needs (Note: this can
be done on the slide warmer or at room temperature. But remember, you
have to determine the correct time and conditions of the stain. For me, the warmer
worked at first, but the older the MacNeals got I realized I had to
stain at RT).

Rinse slide of in water, then place the slide back on warmer, and keep putting
water on, and dumping until the stain leeches to the colors you want. Once at the
proper color, I let dry on the warmer, and mount.

Hope this helps...


Date: 5 Oct 2000 22:43:59 -0500
From: "J. A. Kiernan" <>
Subject: Re: staining plastic-embedded sections

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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