[Histonet] TRAP Assay and IHC

[Adam .]

I'm by no means an expert for cutting frozen mouse bones, but I've done it.
It's certainly more difficult than other tissues, but you can get decent
sections with lots of practice and patience. When I've gotten it to work,
the bones were fixed in formalin or PFA, decalcified in EDTA, cryoprotected
in 30% sucrose, and then snap frozen in OCT using a beaker of 2-methylbutane
sitting in an ice bucket of liquid nitrogen. The knives we use are nothing
special. I don't post-fix after IHC and usually the sections stick to
Superfrost Plus Slides if you let them dry completely before staining.

I'm not entirely sure what you mean by the tissue lifting out of the
section. Sometimes, the marrow can spill out of the bones or the decalcified
bone can fall off and either pull away from the endosteum or even completely
fall off and splay across the section. More often, the periosteum will pull
away from the OCT a bit. I've found the best way to avoid this is to flatten
the section with a paintbrush the best you can, and then quickly and firmly
plant the slide onto the section. I hold the slide on each between my index
fingers and thumbs and slowly lower it until it almost touches. Then I press
down rapidly so the entire section should get pressed onto the slide as
quickly as possible. If you go slowly and allow the section to pull itself
up onto the slide in increments, you'll often get falling off.

I hope that helps,
Adam

On Thu, Feb 4, 2010 at 9:19 AM, Sherwood, Margaret
<MSHERWOOD <@t> partners.org>wrote:

> I want to thank everyone who responded to my inquiry re:  TRAP Assay.  I
> don't
> think I made my request that clear re:  IHC.  We want to do both TRAP Assay
> and
> IHC (separately, of course) on mouse tibia.
>
> We would like to use frozen sections of mouse tibia for IHC.  Our initial
> try to
> cut tibia on the cryostat has resulted in less than optimal sections.  It
> appears that the tissue is lifting out of the section.
> In re:  IHC on frozens -   1)  do you treat the tibia before embedding in
> OCT?
> 2) what knife do you cut with on the cryostat? 3)  do you fix slides with
> cold
> acetone before the IHC procedure (we ususally do)?
>
>
> Thanks again!
>
> Peggy
>
>
> Peggy Sherwood
> Lab Associate, Photopathology
> Wellman Center for Photomedicine (EDW 214)
> Massachusetts General Hospital
> 55 Fruit Street
> Boston, Massachusetts 02114-2696
> 617-724-4839 (voice mail)
> 617-726-6983 (lab)
> 617-726-1206 (fax)
> msherwood <@t> partners.org
>
>
>
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