Re: [Histonet] crumbly femurs

From:"Dana Marshall"

Thanks to those of you have have made suggestions re: my crumbly femurs.  I 
will try to salvage those that i have using these suggestions and be a bit 
more proactive in the future.  Since we do have a cryojane we don't need to 
do the decal so i'll try to get the experiments planned so that people are 
doing that first.  I am happy to hear any experiences with bone so please 
continue to write if you have thoughts on crumbly femurs or good ones!
----- Original Message ----- 
From: "Trajkovic, Dusko" 
To: "Dana Marshall" ; 

Sent: Tuesday, November 08, 2005 3:35 PM
Subject: RE: [Histonet] crumbly femurs

I had a similar experience a few months ago, and it was corrected by
minimally cutting into the surface of the bone or in your case, the
entire side of the leg and placing it in 30% sucrose at 4C for at least
24 hours, then transfer the legs to OCT and let sit for 24 hours at 4C,
before re-freezing in OCT. Since I was doing an Oil Red O on the
sections, and morphology was not an issue, I melted the blocks down,
trimmed the bone as I mentioned above and placed it in sucrose overnight
and then in OCT at 4C. After re-freezing in OCT, bones were sectioning
beautifully, and my ORO worked as well.
Dusko Trajkovic

-----Original Message-----
[] On Behalf Of Dana
Sent: Tuesday, November 08, 2005 12:38 PM
Subject: [Histonet] crumbly femurs

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.
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