neonate skull decalcification without shrinkage

From:Gayle Callis

Use a buffered formic acid, these are slow decalcifiers, approx 4.5% formic
acid, and you can make them up yourself using either sodium formate or
sodium citrate, and also referred to in some publications as acidic
buffers. You can also try a buffered EDTA, disodium salt (takes longer!) at
pH 7.4 - 7.6 or so in TRIS-HCL, there is a reference by Lillie from Stain
Technology on this, or use PBS.  

One thing to remember, the tissue must be TOTALLY fixed in order to protect
the soft tissues and cells from the effects of decalcification. IF you
leave the skulls in an acid decalcifier too long, swelling can occur PLUS
damage proteins via protein hydrolysis from acid.  

If you are going to paraffin process these tissues, you will still get
shrinkange due to dehydration by alcohols, clearing agents and heat of
paraffin, approx 25% and this is unavoidable.   If you have to have NO
swelling or shrinkage at anytime, then it may be better to do undecalcified
bone frozen sections, or fix skulls, then decalcify in EDTA/buffered,
cryoprotect in 20% sucrose (we dissolve this in a buffer), snap freeze and
cut frozen sections. I would not want to cut acid decalcified skulls in a
cryostat, and if you rinse excessively with running water, you can still
get swelling of the tissues.  

 and   At 09:15 AM 10/17/02 -0500, you wrote:
>Hi everyone.  Does anyone have a good way to decalcify mouse skulls that
>doesn't shrink or otherwise alter the brain?  We want to section mouse
>heads from neonates through 2 weeks of age.  I know that we'll have to
>change the time that the brain is in the decalcifying solution,
>depending on the age of the mouse, but if anyone can recommend a good
>decalcifying solution or give any other advice, I'd really appreciate
>Thank you.
>Matt McElwee
>Research Specialist
>Department of Surgery
>K4/617 Clinical Science Center
>600 Highland Avenue
>Madison, WI 53792
>(608) 263-7648
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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