RE: In Situ with Bone

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From:Simon Smith <> (by way of histonet)
Date:Wed, 20 Oct 1999 03:46:51 -0400
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EDTA decalcification is only as painful as you make it.  I have done stacks
of caprine distal femora, decaled in 10% EDTA in 0.1M TRIS (pH 7.0) in 5-10
days.  The trick is to cut your specimens 2-3mm thick, preferably with a
diamond saw.  Have not done in situ, but heard that apoptosis (TUNEL) is
prone to false positives when performed on acid decalcified material.

Undecalcified bone?  Embed in a formulation of PMMA that does not contain a
cross linker, cut sections, stick to slides, dissolve the PMMA and treat as
you would a paraffin section.  The two guys to talk to about this are Neil
Hand and Peter Jackson from the UK.  They have been doing immuno on plastics
for more years than they care to remember and showed some pictures of in
situ performed on PMMA sections at NSH two years ago.

If you need any help, let me know.

Simon Smith
Skeletech Inc
(formerly of NJ, now glad to be in WA where it doesn't rain every

		-----Original Message-----
		From:	Jay Turner []
		Sent:	Monday, October 18, 1999 10:06 AM
		Subject:	In Situ with Bone

		Does any one have any suggestions for a time-efficient in
		hybridization protocol for bone specimens?  I was hoping to
		the long, painful process of EDTA decalcification, if
		Would a dilute formic acid decal solution be gentle enough
for mRNA
		preservation?  Is there an embedding medium for
nondecalcified bone
		that is suitable for in situ?  Any help would be greatly


		Jay D. Turner
		Dept. of Orthopaedics
		University of Medicine and Dentistry of NJ
		Newark, NJ

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