[Histonet] Re: undecalcified bone IHC

[Victoria Baker]

Thank you Gayle.   Vikki
 On Mar 12, 2012 1:04 PM, "gayle callis" <gayle.callis <@t> bresnan.net> wrote:

> Jeff,
>
>
>
> It is most certainly possible to do IHC on undecalcifed bone sections
> embedded in PMMA although not the easiest task.   Sectioning is done on a
> microtome that is powerful enough to cut the plastic and using tungsten
> carbide knives.   The key is total removal of the plastic from MMA embedded
> bone sections to allow antibody/ immunoglobulins to access antigenic sites.
> Neil Hand has done IHC successfully on PMMA embedded tissues including
> undecalcified bone on 2 to 3 µm thick sections.  I think one could cut
> thicker sections at 4 to 5 µm and still be successful.  I do not recall
> what
> Troiano et al used.
>
>
>
> The following publications will help you and should include protocols,
> although conventional protocols will work according to Hand.
>
>
>
> Blythe D. Hand N et al 1997 J Clin Path 50:45-49.    The use of methyl
> methacrylate resin for embedding bone marrow trephine biopsies.
>
> Hand NM et al 1996 Antigen unmasking using microwave heating on formalin
> fixed tissue embedded in methyl methacrylate J Cellular Path 1:31-37
>
> Jackson P et al.   1996  Amplification of immunocytochemical reactions by
> the catalytic deposition of biotin on tissue sections.   J Path
> 170(suppl):23A.  This was about tyramide amplification when one gets a weak
> signal from "conventional" methods.
>
> Hand NM, Church RJ 1998 Superheating using pressure cooking: its use and
> application in unmasking antigens embedded in methyl methacrylate.  J
> Histotechnology 2`:231-236
>
> Hand NM et al 1989 Immunohistochemistry on resin embedded tissue for light
> microscopy: a novel post embedding procedure.  Proceeding Royal
> Microscopical Society 24(1):A54-55.
>
> Hand NM Plastic Embedding media and techniques, Ch.30, p 663-677.   Theory
> and Practice of Histological Technique,  5th edition by Gamble and
> Bancroft.
> The 6th edition is updated under same title.
>
>
>
> Use Google Scholar to find Troiano N et al from Yale on doing IHC on PMMA
> embedded bone sections with publications in J Histotechnology.
>
>
>
>
>
> Hand mentioned several HIER methods, using citrate buffer.   Optimizing
> retrieval will depend on the antigen and you may end up doing this with
> some
> form of HIER, including microwave or other heat producing methods and with
> different buffers.     Enzyme digestion is also a possibility.
>
>
>
> Hand removed MMA with xylene, warm my speed up the removal, also more than
> one change for 10 - 20 minutes or longer.   When I talked to him
> personally,
> he said he had used warm xylene although temperature was not mentioned in
> his chapter.   After MMA removal, rehydrate section through alcohol
> gradient
> as one does paraffin sections.    He was emphatic about never allowing the
> sections dry out.
>
>
>
> Hopefully Jack Ratliff and Damien Laudier will provide more insight on this
> topic.
>
>
>
> Good luck
>
>
>
> Gayle M. Callis
>
> HTL/HT/MT(ASCP)
>
>
>
>
>
>
>
>
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>
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>
>
> ******************************************
>
>
>
> Hi Jeff,
>
>
>
> If is it possible a few more specifics of how the tissue has been received,
>
> processed and evaluated would help.  Undecalcified bone sectioning
>
> procedures vary and also what specific markers are you looking to do is
>
> important.
>
>
>
> Vikki
>
> On Mon, Mar 12, 2012 at 11:06 AM, Rene J Buesa <rjbuesa <@t> yahoo.com>
> wrote:
>
>
>
> > Undecalcified? How are you going to section it?
>
> > If you can section it, just use any IHC protocol for regular sections.
>
> > Good luck!
>
> > René J.
>
> >
>
> > --- On Mon, 3/12/12, Jeffery Howery <Jeffery.Howery <@t> jcl.com> wrote:
>
> >
>
> >
>
> > From: Jeffery Howery <Jeffery.Howery <@t> jcl.com>
>
> > Subject: [Histonet] Undecalcified bone IHC
>
> > To: histonet <@t> lists.utsouthwestern.edu
>
> > Date: Monday, March 12, 2012, 10:59 AM
>
> >
>
> >
>
> > Does anyone have a protocol for Undecalcified bone for IHC?
>
> >
>
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