[Histonet] suggestions on bone cross section?

[Gayle Callis]

I presume mouse you are working with mouse?   Are the bones prefixed?   If 
so, you will need to sucrose cryoprotect before snap freezing.  You will 
need a tungsten carbide knife, pricey!! and I strongly advise buying 
two.  One in use, one being reconditioned at $220 a knife.  We use D 
profile tungsten carbide tipped cryostat knives from Delaware Diamond 
Knives, and prefer these over the C profile tungsten carbide.

Bones can be laid flat in an plastic embedding mold from Tissue Tek, embed 
inOCT, then snap freeze, preferrably with dry ice mixed with hexane.  All 
you have to do for precise oreintation, is take frozen block, turn it on 
end with area you want to see facing up, and mount block on a metal 
cryostat chuck.  Be sure to surround block with more OCT at this time to 
build up block face.   If you mount the block with 2% methyl cellulose, you 
will have a very hard, sturdy support of the block but do not use this goo 
to embed with, it is horrible to cut.

You can even stand bone on end inside a peel a way mold, the 22 mm x 22 
mm.  Hold the bone while OCT starts to freeze, then let it totally 
freeze.  You now have a block that is long enough with a big enough block 
face to cut nicely.  This amount of OCT will freeze more slowly, and you 
may get freezing artifact.  You can freeze bones first (coated with just 
diluted OCT 1:1 with PBS, then double embed the frozen bone in this mold 
with more OCT,  using dry ice/hexane slurry.  You need to avoid warming up 
already frozen bones, use forceps that are stored in slurry to hold bone 
until bottom of mold begins to freeze.  Be sure to release forceps before 
block is totally frozen.  This will be easier than trying to stand a frozen 
bone upright on a chuck and add more OCT.

We set the cryostat temperature to -29C or colder with knife and sample 
also that cold.

Good luck as cryosectioning of tiny cortical bones will NOT be easy.



Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)