Marking ink for maintaining orientation RE: [Histonet] freezing mouse eyes

[Gayle Callis]

I agree that orientation is always a problem with small samples.  We have 
used green (color is easy to see) marking ink to maintain orientation of 
samples for snap freezing while using the petri dish floating in liquid 
nitrogen snap freezing method.  Care must be taken NOT to be heavy handed 
with the dye as too much will defeat orientation purpose when a tiny sample 
is completely smeared with green!    An extremely fine brush helps with dye 
application along with some type of magnifier -  we use eye glass loupes 
attached to our spectacles.  Ink can be used on any of the type of samples 
you mention.

Using very fine forceps, one can orient the eye to bottom of plastic Tissue 
tek cryomold as the OCT begins to freeze (turns white)  - with practice, it 
can be done.  We do this with cross sections of very tiny murine spinal 
cord and maintain orientation without problems.  Eyes are larger in 
diameter than some of the cord cross sections.   With practice, one should 
be able to maintain orientation with any type sample using careful inking.

At 02:13 PM 11/22/2004, you wrote:
>I would ask what the investigator hopes to learn from the frozen specimens,
>and how they would like that accomplished. I have done staining both
>chemical and immuno on fresh frozen, fixed  cryoprotected frozen, and
>paraffin embedded mouse eyes.  I find the most difficult area to be the
>orientation for embedding.
>
>Cynthia Favara
>NIAID/NIH/RML/LPVD
>903 South 4th Street
>Hamilton, MT 59840
>406-363-9317
>
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>-----Original Message-----
>From: Danielle Crippen [mailto:dcrippen <@t> buckinstitute.org]
>Sent: Monday, November 22, 2004 11:24 AM
>To: histonet <@t> lists.utsouthwestern.edu
>Subject: [Histonet] freezing mouse eyes
>
>Dear all,
>
>I have several sets of mouse eyes from perfused mice which have been in 4%
>PFA over the weekend.  The investigator has just informed me this morning
>that she needs these eyes frozen.  Can anyone with experience please
>elucidate what you would do in this scenario??  Should I cryoprotect?  If
>so, should I use a sucrose gradient: 10%, 20%, 30%??  Also, how long should
>each of these steps be??  At our institution, the most common freezing
>method is by floating the tissue in a foil boat in liquid nitrogen.  Any
>better suggestions??
>
>Many many thanks to all the experts who I'm hoping will respond:-)
>
>Cheers,
>
>Danielle Crippen
>
>
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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)