Re: undecalcified bone (Cryosectioning)

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From:"R.Wadley" <>
Date:Fri, 04 Jun 1999 08:37:24 +1000
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	Dear All,

	I have only been following this cutting undecalcified bone discussion with
1/2 an eye, but I thought I might add my experience.

	In a previous position I had to section whole mouse legs to determine the
infiltration (into muscle/bone) or otherwise of a fluorescent dye that had
been injected prior to sacrifice of the animal.  I used a standard Leica CM
1800 cryostat and disposable knives.  Initially I used standard low profile
knives but I was much more impressed by the results when I used gold coated
high profile blades by Sturkey.  I'm sure most other high profile blades
designed for cryo work would be as good.  

	Generally speaking the most important factor when working in a cryostat is
the temperature.  My notes suggest somewhere between -23 to -30C.  I almost
never even considered changing the knife angle unless I had a special case
of something particularly unusual.  (This was a research lab where we cut
anything from hard & soft tissue to gels.)  I haven't tried cryo tape on
small sections, but had a lot of success with it when I was sectioning
whole mice (less head, limbs & tail) at ~60 um.  I just rang my local tape
wholesaler & asked for something that sticks in the cold.  I had the choice
of two, 1 clear & 1 silvered.

	It is a mistake to get bone too cold, especially cortical bone.  I was
cutting muscle-bone-muscle, so had to make some compromises.  So this might
be a case of if it doesn't work at -30C then try warmer temps rather than
colder ones.

	Hope this helps.



At 08:49 AM 6/3/99 -0600, you wrote:
>Even if you put murine bone in polymethylmethacrylate, you will get 25%
>shrinkage, which is comparable to paraffin (have a publication on this,
>a comparison!)  Bone frozen sections are easiest done with an
>Instrumedic Cryojane Tape Transfer system on snap frozen bone and you will 
>need a tungsten carbide d profile knife.  Both are expensive, sectioning
>system is approx $7000 and knives from DDK are approx $950, not to mention
>a cryostat.  
>However, the results are well worth the investment, and saves a good deal
>of grief.

R. Wadley, B.App.Sc, M.L.S
Laboratory Manager
Cellular Analysis Facility
School of Microbiology & Immunology
UNSW, New South Wales, Australia, 2052
Ph (BH) 	+61 (2) 9385 3517
Ph (AH)	+61 (2) 9555 1239
Fax 	+61 (2) 9385 1591
	(Under development)

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