Re: [Histonet] crumbly femurs

From:Gayle Callis


You did not say that you cryoprotected the fixed/decalcified mouse bones in 
30% sucrose at 4C for a few days.  If not, you don't get very good frozen 
sections.  Then you should section at -26C with Cryojane, even with 
decalcified/cryoprotected.  I'm a bit surprised that Cryojane is not 
helping you.

It also helps to use a high profile blade or even use a c profile tungsten 
carbide for doing decalcified free hand bone FS,  much sturdier 
knives/blades for denser bone.

Did you do endpoint determination during decalcification? The crumbly bone 
may be in indication your bones are NOT completely decalcified - a disaster 
for frozen sections.  If you are doing frozen sections with Cryojane in the 
first place, why do you need to have bones fixed and decalcified?  I 
suggest you try undecalcified, snap frozen FRESH bones instead but make 
sure the tungsten carbide knife d profile is really sharp. Some use c 
profile, we prefer d profile.

If you did EDTA decalcification, they bones sometimes take more than a week 
to be totally calcium free.

At 01:37 PM 11/8/2005, you wrote:
>Hi everyone,
>I am trying to section some mouse legs.  The bones were fixed/decalcified 
>for one week then quick frozen in OCT and put at -20C where they have been 
>for about 2 months.  Regardless of how thick I cut the section (5-20 
>microns), whether I cut free hand, use our cryojane or the old fashioned 
>scotch tape type of tape transfer, there is little/no visible bone in the 
>section.  Just OCT and an empty space where bone would be.  If I cut 
>without tape I can see the bone crumble as I cut.  This seems to be a 
>catastrophic failure of some sort and may be beyond any type of help at 
>this point but if anyone has any ideas as to why this is happening I would 
>be happy to hear from you.
>Histonet mailing list

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)

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