Re: [Histonet] Cells Embedded in Collagen for Frozen Tissue Sectioning (Omer Richman)
You did not say what temperature you were cryosectioning at? But if the
temperature is too cold and the collagen is separating from the little
ball, then warm up the temperature with the same temperature for both block
and blade. A perfectly round ball i.e your collagen balls have always
been more difficult to handle so they do not curl at sectioning. We found
this out in our lab too. Whya that little ball curls must be some
mechanical problem due to the round shape versus a shape with some type of
point on it. We did agar type balls both as frozen sections and paraffin
sections and both curled up and out of the both types of embedding medias.
You may want to try a different cryoembedding media, Thermo Electron has
one which is supposed to work nicely for colder temperatures. Andy
Grantham and Teri Johnson have used this one - it may help, and it was
recently mentioned by Andy in a Histonet messAGe. Also when embedding,
make sure you blot the excess sucrose off and let the balls sit in OCT for
a short time before snap freezing - you may get a better interface of
collagen to embedding media.
Another alternative to keep the balls from curling is using the
Instrumedics tape transfer Cryojane - but not everyone has this instrument,
but the tape would keep the balls from curling up.
Good luck on solving your frustrations
Gayle Callis HTL, HT, MT(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717
At 07:37 PM 11/14/2006, you wrote:
>I apologize for leaving out some critical elements, I had only a few moments
>to type that up. The cells are pre-fixed in the collagen prior to embedding
>and we do wait until the collagen sinks (generally overnight). The cells are
>in the center of the collagen in a ball. Prior to freezing in OCT, it's easy
>to see them but once it's in the OCT block I find it difficult to
>distinguish them from the rest of the collagen. Also, the collagen separates
>from the rest of the OCT during sectioning so that, even if I could find the
>cells, I feel I may lose them during the act of cutting. That leads me to
>believe that perhaps I'm not using the right temperature. At what
>temperature do you normally cut collagen?
>Gayle's answer actually helped in a related experiment so I extend my thanks
>to her for that. However, my PI reminded me we need to use collagen because
>we want to see how far the cells migrate then follow that up with IHC. Thank
>you all again for your assistance.
>State University of New York at Stony Brook
>Life Sciences Building Rm 004
>Stony Brook, NY 11794
>On 11/14/06, MVaughan4@ucok.edu wrote:
>>You don't mention whether you fix the cells in the collagen ahead of
>>embedding. Also, do you infiltrate the collagen gel in 30% sucrose in PBS
>>until the collagen sinks? That has always been our protocol and we had no
>>problem sectioning collagen gels. I guess the bigger problem is, where
>>are the cells in the collagen? And it looks like Gayle has a better answer
>>than blindly sectioning collagen.
>>Melville B. Vaughan, Ph. D.
>>Department of Biology
>>University of Central Oklahoma
>>100 N. University Drive
>>Edmond, OK 73034
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