Re: JB4 aka GMA plastic staining

From:Gayle Callis <>

The best article I have read on plastic, GMA and PMMA, discussed this and
stated GMA as being LESS hydrophobic than PMMA.  It was written by Horobin
and had indepth discussion about how and why plastics stain, with what
molecular weight dyes successful on what plastic, temperature, pH and
plastic removal considerations. Wonderful article.  

GMA allows high and low molecular weight dyes, aq and otherwise to stain,
PMMA works with low molecular weight dyes (t blue) but will not stain with
alcian blue UNLESS the MMA is totally removed. 

Interesting, if GMA was really hydrophilic, wouldn't it redissolve in water
(wouldn't that be sweet, in order to remove it) after it is polymerized.
Instead it just softens, swells but remains intact, polymerized!!

By the way, Jay Beckstead is now a practicing pathologist in Southern
Oregon, he made some wonderful contributions to our field, particularly the
zinc TRIS buffer fixative (non formalin) for CD marker staining/paraffin


>From: "Thomas J. Kuwahara" <>
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>To: Gayle Callis <>
>Subject: Re: JB4 aka GMA plastic staining
>Hi Gayle!:  Just a note to follow up on Gayle's observation of IHC
>staining on JB4.  I used to do some limited staining at SF General
>Hospital using Dr. Jay Beckstead's method.  It involved cutting sections
>onto glass cover slips and lots of o/n incubations.  It worked sort of
>but was very faint most of the time.  I also found that as time went on
>the blocks lost their antigenicity as well.  Only the freshly  cut
>sections seemed to work.  Storing the blocks in the freezer only
>partially helped.  After Dr. Beckstead left UCSF, I gratefully and
>immediately stopped doing the procedure.  Definitely not worth the time
>or effort for IHC.  Correct me if I'm wrong,Gayle, but isn't JB4
>hydrophilic (MMA is hydrophobic?), or else it wouldn't take up any
>aqueous staining at all?
>Regards, Tom
>> You can use most stains performed on paraffin sections, with the exception
>> of some trichromes, they tend to be more difficult.  However, there is a
>> modification of this stain for 1 um sections that work beautifully.
>> You can do Jones Methenamine silver, Giemsa, H&E, etc.  One thing that is
>> nice the plastic does not (cannot!) be removed, and excessive dehydration
>> in alcohols, clearing in xylene will cause plastic to crack.  We usually
>> air dried after staining, and coverslipped a DRY section with routine
>> mounting media.
>> Eosin Phloxine counterstain is wonderful, can buy this from Richard Allan
>> or make up yourself.
>> Immunostaining is more difficult often not possible since plastic tend to
>> prevent immunoglobulins (hydrophobic problem) access to antigenic epitopes.
>> Enzyme histochemistry can also be performed, plus some technics for
>> embedding and lipid staining.
>> Take care, this stuff is toxic, work in a hood, with nitrile gloves, wear
>> safety glasses when sectioning (heard of a section flying into technicians
>> eye) keep skin contact down to very minimum, never with wet hands.  Very
>> sensitizing material, plus carcinogens in accelerator.
>> There are publications on GMA in Journal of Histotechnology, back in 70's
>> Clyde Lulham publication and Nate Brinn on enzyme histochemistry, plus
>> others over the years.
>> Gayle Callis
>> Veterinary Molecular Biology
>> Montana State University
>> Bozeman MT 59717-3610
>> 406 994-4705
>> 406 994-4303
>Thomas J. Kuwahara - Senior Immunohistochemist
>Resolution Sciences Corporation -
>3801 Sacramento St., Suite 621, San Francisco, CA  94118
>T: 415/750-6800, ext23067 F: 415/750-2332  E:
Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303

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