Sharon,

It's my understanding that the tetracycline and other fluorochromes 
used in this application (xylenol orange, calcein, etc) bind to the 
calcium in bone.  Therefore, if you decalcify, you are eliminating 
the signal, regardless of what technique you subsequently use.  Can 
you section this stuff without decalcifying?  If you can, my bets are 
that the rings will be there.

-Karen








>Date: Tue, 30 May 2000 10:38:35 +0000
>From: "Marshall, Sharon," <marshall@anat.uct.ac.za>
>Subject: ageing rings in sharks
>To: histonet@pathology.swmed.edu
>
>Hi histonetters,
>I have a student in my lab. who wants to view ageing rings in cat
>shark vertebrae.  This has been done before (previous papers) using
>resin sections and tetracycline fluorescence.  These sections are a
>couple of mm thick and viewed under ultra violet light. She seems to
>think it might be possible to process to wax, cut 7 micron thick
>sections and still see these rings.  We tried some samples she had
>which are 10 years old and have been in 70% alcohol all that time.(no
>tetracycline present in sharks in these samples)
>We decalcified in 5% formic acid, processed to wax and then stained 7
>micron sections with H&E, Tol. Blue. No rings.  I think these rings
>are only possible to view using thicker sections i.e.mm thick or the
>specimens are to old.   I know that to see tetracycline flourescence
>one needs to alcohol fix and resin embed hence the processing in the
>previous papers.
>Finally, we are going to try fresh samples but I am wondering if we
>are not wasting our time.  I need to convince her that resin and
>thicker sections cut with a diamond saw is the
>way to go even if no tetracycline is present in the samples.
>Any opinions,help would be great.
>Thanks
>Sharon Marshall
>Anatomy & Cell Biology
>University of Cape Town
>South Africa
>E-mail: marshall@anat.uct.ac.za