[Histonet] More details: decalcified sections of bone

Nicole Hedgecock n_hedgecock <@t> hotmail.com
Tue Oct 5 16:44:50 CDT 2004

I sent this out earlier (see below) and more details of my tissue processing 
and sectioning were requested.
I want to identify apoptotic osteocytes in rabbit cortical bone as well as 
some morphological features especially microcracks.  I fix the bones in 4% 
paraformaldehyde for 24 hours, then place then in 70% EtOH until I can 
decalcify them in EDTA at room temp.  Between the EtOH and EDTA steps I 
rinse the bones in dH2O for about 3 days.  After decalcification, the bones 
are again rinsed in dH2O and then embedded in paraffin and sectioned to 6 
microns.  I use the TUNEL kit from Roche for In situ labeling of DNA breaks.
Do you think that I have to do decalcification for the TUNEL to work? Does 
sectioning of paraffin embedded tissue create a lot of microscopic damage to 
the section?

I hope this clears up my situation and goals.

>From: "Nicole Hedgecock" <n_hedgecock <@t> hotmail.com>
>To: histonet <@t> lists.utsouthwestern.edu
>Subject: [Histonet] decalcified sections of bone
>Date: Tue, 05 Oct 2004 14:42:45 -0400
>Hi, All
>I am attempting to do TUNEL staining of osteocytes in cortical bone.  I was 
>wondering why the bone has to be decalcified in order to do this process.  
>And Why do the sections have to be so thin?
>Nicole Hedgecock
>Master's Student
>Orthopedic Research Lab
>UCD Medical Center
>University of California, Davis
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