[Histonet] Re: undecalcified bone IHC

Tue Mar 13 10:55:44 CDT 2012

Excellent! Happy to read the venerable Neil Hand is coming back to the
symposium, always a great speaker!

-Damien

On Tue, Mar 13, 2012 at 10:50 AM, Jack Ratliff <ratliffjack <@t> hotmail.com>wrote:

>
> I might also add that Neil Hand is co-speaking with myself and Philip
> Seifert this year at the annual National Society for Histotechnology -
> Symposium/Convention in Vancouver B.C. Our workshop is titled:
>
> Resin Applications Forum: Methods for Processing, Special Staining,
> Immunohistochemical and In Situ Hybridization of Soft and Hard Tissue
> Including Medical Device Implants
>
> During the last 50 years, numerous histological procedures have been
> described on resin embedded tissue. While different types of resins are
> available for different purposes, the acrylics provide the widest range of
> techniques, especially for light microscopy applications. However, as
> demand from H&E to more sophisticated techniques increases, so too have the
> problems, and nowhere is this more apparent and controversial than in the
> application of immunohistochemistry on resin sections. This workshop will
> provide a review and discussion for those individuals that currently work
> with and/or are just getting started working with soft and hard tissue
> specimens and specifically the various resins (i.e. MMA, GMA, Technovit,
> Acrylosin, etc.) associated with their specific tissue interests. The
> workshop will also detail the preparation and staining of sections of soft
> and hard tissue, including implants (e.g. undemineralized bone and
> cardiovascular stents), for immunohistochemical and in situ hybridization
> staining using different acrylic and epoxy resin embedding media. Specific
> problems and pitfalls, either technical or operational associated with
> certain resin embedding procedures, will be illustrated and examined.
> Particular emphasis will be given to procedures which have been used
> extensively for routine diagnostic, and research purposes, i.e. those that
> WORK! Individuals with a current or future intent to process and cut
> undemineralized tissue or tissue containing foreign implant materials using
> acrylic or epoxy resins are strongly encouraged to attend this workshop!
>
> Please feel free to contact me if you would like more information about
> the workshop as information relevant to the exact date and time becomes
> available. All I know at this time is that the NSH meeting is September
> 29th - October 3rd, 2012.
>
> Best Regards,
>
> Jack
>
>
> Jack Ratliff
> Hard Tissue Histologist
> Chairman, Hard Tissue Committee - National Society for Histotechnology
>
>
>
>
> > From: gayle.callis <@t> bresnan.net
> > To: histonet <@t> lists.utsouthwestern.edu
> > Date: Mon, 12 Mar 2012 11:04:20 -0600
> > Subject: [Histonet] Re: undecalcified bone IHC
> >
> > 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)
> >
> >
> >
> >
> >
> >
> >
> >
> >
> >
> >
> >
> >
> > ******************************************
> >
> >
> >
> > 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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>
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-- 
Damien Laudier
Laudier Histology
www.LaudierHistology.com