[Histonet] RE: embryo cryosection problem

[Gayle Callis]

Cryoprotection in 25% - 30% sucrose should improve your sectioning.  We 
make ours up in Dulbeccos PBS, and let the fixed embryos sit in these 
overnight at 4C, but yours are older at dpc 13 - 17, they could sit longer.

Depending on what you are doing (you did not say) decalcification with EDTA 
is an alternative but you must still cryoprotect after decalcification if 
you still see minute boney parts ripping out.  You could use a tungsten 
carbide c profile blade to help deal with these minute fragments IF you 
didn't want to decalcify.

Good luck


At 07:15 AM 11/14/2004, you wrote:
>hi all,
>
>    I recently tried cryosectioning embryos taken at dpc13-17, and had a
>horrible time because sections were crumbling to pieces, due to what I
>believe was some really 'crusty' bone or cartiledge sticking out and
>ripping the tissue as the blade passed over it (this is in the head).
>I am new to working with embryos, and I am trying to figure out what I
>did wrong!  I fixed embryos 24-48 hours in 4% paraformaldehyde, and
>then snap froze in OCT.  should I have decalcified these specimens?  or
>cryo-protected them?  I would greatly appreciate anybody's advice on
>this!  thanks so much in advance!
>
>Ana Beaudin
>Division of Nutritional Sciences
>Cornell University
>Ithaca, NY
>
>
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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)