RE: ossification long answer includes procedure

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From:"Anita Jennings" <>
To:"'Gayle Callis'" <>, <>
Date:Fri, 27 Aug 1999 11:40:23 -0700
Content-Type:text/plain; charset="iso-8859-1"

I almost put together a poster on this very thing for this years NSH
symposium......maybe next year. Like you said they look really impressive
and there are a few tweaks that need to be shared to make it easier to do.
We use two different procedures depending on the age of the mouse. If you
decide to embed for sections that is a "whole nother paper".
The one is from the back of "The Atlas of Mouse Development" M.H.. Kaufman
(bet you have this book)
1L stain solution:
75 ml Alizarin Red (0.1% in 5% acetic acid)
50 ml Alcian Blue (0.05% in 5% acetic acid)
50 ml Acetic Acid
825 ml 100% EtOH

For newborn mice (evisceration not necessary for embryos <15.5 dpc)
1. Remove skin from mice and eviscerate. Do not attempt to remove the heart
if the rib cage is necessary
2. Fix the mice in 100% EtOH, 4 days without shaking
3. Post fix mice in acetone, 3 days with shaking
4. Stain embryos in staining solution, 5 days with shaking
5. Rinse embryos with water and transfer to 20% glycerol/1% KOH, 16 hours at
37C. Move to room temperature until tissue is completely cleared.
6. For storage, run skeletons through a glycerol series/1% KOH: 50%, 80%
7. Photograph using dissecting microscope.
 The other reference comes from "Manipulating the Mouse Embryo A Laboratory
Manual" Hogan et al Cold Spring Harbor. It is a little more drawn out but I
can send you a copy if you want. Good luck call if you need more specifics.

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