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

[Gayle Callis]

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 
hexane fumes.

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