[Histonet] Re: undecalcified bone IHC

[gayle callis]

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?

>