Apart from the obvious step of using a higher concentration of enzyme, you
could try a mixture of trypsin and pepsin. Also, formaldehyde fixation can
be largely undone by extended washing. Try washing the tissue in running
water for 24 hours or longer, before doing the digestion procedure.

> ----------
> From: 	histonet-bounces@lists.utsouthwestern.edu on behalf of
> Boyce, Amanda (NIH/NIAMS)
> Sent: 	Thursday, August 25, 2005 7:30 AM
> To: 	'histonet@lists.utsouthwestern.edu'
> Subject: 	[Histonet] muscle digest for sekeltal prep
> 
> We are processing animals for skeletal preps that have been in 10% PFA for
> quite some time (many months).  The first steps involved skinning and
> eviscerating the embryos, followed by alcian blue staining.  We then put
> the
> embryos in a trypsin digetion solution, which normally chews up the
> majority
> of the muscle.  The rest of the muscle will be dissolved after the
> skeletons
> are stained with alizarin red and put through a series of KOH washes.  The
> problem is that because these animals are overfixed, the trypsin digest is
> not sufficient.  Does anyone have any suggestions for a more vigorous
> digestion?  Thanks for your help!  Amanda
> 
> --------------------------
> Amanda Taylor Boyce, Ph.D.
> Postdoctoral IRTA Fellow
> Cartilage Biology and Orthopaedics Branch
> National Institute of Arthritis and Musculoskeletal and Skin Diseases
> National Institutes of Health
> Bldg. 13, Rm. 3W17
> Bethesda, MD 20892-5755
> Phone: 301-451-6860
> Fax: 301-480-4315
> Email: boycea@mail.nih.gov
> 
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> 

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