Re: Processing Intervertebral Disks

From:Gayle Callis

Your samples are fairly large, fixation sounds good hopefully with a change
of NBF after a couple of days to replenish fixing agent. You can speed up
the decalcification by using 10 - 15% formic acid with your totally fixed
bones, but be sure you do endpoint testing to insure calcium is removed and
avoid overexposure to acid, preferrably x ray technics.  

You did not say what species you are working with?  I think you need to
extend your processing times to 3 hours per station including the
paraffins, with 3 changes minimum there under vacuum.  I do whole rat knees
and femurs at 2 hours per station. Schedule that worked for us ethanols,
70%, 80%, 95% X 2, 100% x 2, xylene X 2 (or xylene X 1, clearite 3 X 1 -
improves cutting) paraffins X 4, or 3 minimum  We also use Tissue Prep 2, a
harder paraffin for infiltration and embedding of bone. Ambient
temperatures throughout, with vacuum and pressure on.  We have evne used 4
hours per change with success for really huge, denser bones. 

If you have to, melt away paraffin, go back into clearing agent two changes
for couple of hours each, and put into 100% alcohol, couple of changes to
remove all water, then reprocess into clearing and paraffins with longer
times.  You might be able to salvage the soft, poorly infiltrated and
possible poorly dehydrated specimen. You do not have to go all the way back
to 70, 80 or 95%.  
Good luck,

At 10:54 AM 7/17/02 -0500, you wrote:
>Hello Histonetters,
>I am having some problems processing en block intervertebral disk specimens 
>(flanked by portions of vertebrae).  I am especially interested in 
>examining the nucleus pulposus.  The samples are about 1 cm thick, fixed 
>for about 10 days in 10% formalin and then decalcified in 5% formic acid, 
>anywhere from 1 to 1.5 weeks.  The processing schedule I am using is 1.5 
>hours in each of the dehydrating, clearing, and paraffin stations.  After 
>coarse trimming to the area of the nucleus pulposus, I am finding that this 
>portion of the paraffin block is soft like is was improperly infiltrated 
>and/or processed.  For the most part, the anulus fibrosus is intact except 
>at the nucleus pulposus interface.
>Is this simply a matter of increasing the processing times or is there 
>something else I am neglecting to do?  Any help would be greatly appreciated!
>Gratefully yours,
>Tracey Couse
>Laboratory Coordinator/Histologist
>Georgia Tech/Emory Center for the
>    Engineering of Living Tissues
>Georgia Institute of Technology
Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)


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