Fresh frozen bone sections

From:Gayle Callis

You will find this discussed in Histonet archives, but we do bone frozens,
snap frozen with a dry ice/hexane slurry (RA Dodds method), and cut in a
cryostat commonly found in clinical work, Cryocut 1850, at -30C using a
tungsten carbide knife, d profile preferred over a c profile Tungsten
carbide, and the Instrumedic tape transfer Cryojane system, with 1/2X
polymer coated slides.  Our D profile cryostat knives were made via special
order from DDK since we did not like the C profile TC cryostat knives as

GFP is a whole other story, some have more success than others, but pH
extremes can denature the protein, ruining its autofluorescence.  Some
people have had success with decalcification/paraffin work, but others seem
to have poor to no results.  

I will be happy to discuss bone frozen further if you wish. 

At 05:36 PM 7/16/02 -0700, you wrote:
>Hi there,
>we are facing a problem preserving GFP (EGFP, DsRed, Clontech) visualization 
>in bone sections. To our knowledge we have to perform a decalcification.
>anybody has experience with a protocol leaving GFP visualization intact ? 
>Should we use EDTA, formic acid ? In which concentrations ? Preferably we 
>would like to perform cryosections rather than parraffin processing. Does 
>anybody has experience with a method preparing fresh frozen sections of 
>undecalcified tissues (Kawamoto T. and Masaharu S. Histochem Cell Biol 2000, 
>113: 331-339).
>Any help on this problem would be very appreciated.
>Martin Weber
>The Scripps Resaerch Institute
>10550 North Torrey Pines Road
>La Jolla, 92037 C.A.
>Phone: 858-784-8612
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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