[Histonet] crumbly femurs

[Alan Bright]

Dear Dana,

Looks to me that your specimen has freeze dried due to being stored  @  -20ºC for 2 months. I agree that you should have no problems sectioning fresh frozen undecalcified mouse legs with a tungsten carbide tipped knife, but do not see the need for using any tape removal system, just a good cryostat and anti roll plate sectioning @ -30ºC with a slow cutting speed.


Best Regards

Alan Bright

Bright Instrument Co.Ltd.
St Margaret's Way
Huntingdon
Cambridgeshire
PE29 6EU
England

Tel No:+44 (0)1480 454528
Fax No:+44 (0)1480 456031
Email: abright <@t> brightinstruments.com
Web Site: www.brightinstruments.com



-----Original Message-----
From: Gayle Callis [mailto:gcallis <@t> montana.edu] 
Sent: 09 November 2005 15:44
To: Dana Marshall; Histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] crumbly femurs


Dana,

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.
>Thanks,
>Dana
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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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