[Histonet] Snap freezing undecalcified bone using hexane, OCT ingredients

[Gayle Callis]

At last, an answer!  Thank you.  But on the other hand, when freezing whole 
rat femurs, skulls, or where the bone marrow is not exposed and totally 
surrounded by cortical bone, I guess one could not bother with coating the 
bones.  Wet spongy centers are something I have not experienced when I 
haven't coated bone but then these were whole samples dissimilar to some of 
his samples (ostephytes cut off from larger bones).

At 11:43 AM 11/27/2006, you wrote:

>To answer one of your questions in your e-mail, Dodds told me that he 
>coats the bone with PVA so that the hexane is not wicked into the bone 
>marrow. If this happens, you wind up with a wet spongy center.
>
>Jeff
>-------------- Original message from Gayle Callis <gcallis <@t> montana.edu>: 
>--------------
>
>
> > We do this all the time, and it can be done two ways.
> >
> > RA Dodds was one of the first who used this along with van Noorden. Dodds
> > published in J of Histotechnology. Check out the following references for
> > more on snap freezing bone, particularly the van Noorden paper. Hexane is
> > one solvent used and it seems to be a gentler but thorough snap
> > freezing. There is extensive discussion about snap freezing bone and the
> > various solvents in Histonet archives.
> >
> > JR Connor, RA Dodds, IE James, and M Gowen
> > Human osteoclast and giant cell differentiation: the apparent switch from
> > nonspecific esterase to tartrate resistant acid phosphatase activity
> > coincides with the in situ expressi! on of osteoponti n mRNA
> > J. Histochem. Cytochem., Dec 1995; 43: 1193 - 1201.
> >
> > RA Dodds, K Merry, A Littlewood, and M Gowen
> > Expression of mRNA for IL1 beta, IL6 and TGF beta 1 in developing human
> > bone and cartilage
> > J. Histochem. Cytochem., Jun 1994; 42: 733 - 744.
> >
> > CJ Van Noorden, IM Vogels, and RE Smith
> > Localization and cytophotometric analysis of cathepsin B activity in
> > unfixed and undecalcified cryostat sections of whole rat knee joints
> > J. Histochem. Cytochem., May 1989; 37: 617 - 624.
> >
> > Dodds dipped undecalcified bone in a water soluble 30,000 - 70,000 MW
> > polyvinyl alcohol to coat the bone, and then snap froze in a mixture of 
> dry
> > ice and hexane (pieces of dry ice in the hexane itself). We have done this
> > but coated the bone with diluted OCT (1:1 OCT 1:1 with water since the OCT
> > contains polyvinyl alcohol. Dodds never explained why coating was > 
> important unless to protect the surface of the bone from some of the
> > effects of the solvent. The frozen bone could then be attached to the
> > metal disk with either water, OCT, or some other cryoembedding media (2%
> > methyl cellulose is a super hard hold for bone).
> >
> > OCT contains polyvinyl alcohol, polyethylene glycol and water. The
> > molecular weights of these chemical are given, a proprietary issue, but we
> > use it for undecalcified bone cryotomy along with the Instrumedics 
> Cryojane
> > tape transfer system and have good results.
> >
> > We prefer to embed the bone in OCT, then snap freeze with the hexane/dry
> > ice mixture. Hexane is explosive, and not a good thing to breathe
> > in. Fume hoods should be used and some use respirators to protect from
> > hexane fumes.
> >
> > One thing that can happen with liquid nitrogen snap freezing is bone
> > shatters or splits. We exper! ienced this with immature bovine bone and 
> also
> > delicate nasal bones on a mouse head which will split apart. The liquid
> > nitrogen temperature may be too cold for this calcified matrix but
> > hexane/dry ice freezing prevents this shattering.
> >
> >
> > Gayle Callis HTL, HT, MT(ASCP)
> > Research Histopathology Supervisor
> > Veterinary Molecular Biology
> > Montana State University
> > Bozeman MT 59717
> >
> >
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