[Histonet] Snap freezing undecalcified bone using hexane, OCT ingredients
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 expression of osteopontin 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
One thing that can happen with liquid nitrogen snap freezing is bone
shatters or splits. We experienced 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
Histonet mailing list
<< Previous Message | Next Message >>