[Histonet] Snap freezing undecalcified bone using hexane,
gcallis <@t> montana.edu
Mon Nov 27 14:45:48 CST 2006
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.
>-------------- 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
> > 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
> > 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
> > 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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