Re: [Histonet] Cells Embedded in Collagen for Frozen Tissue Sectioning (Omer Richman)

From:Gayle Callis


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.
>Omer Richman
>Research Technician
>State University of New York at Stony Brook
>Life Sciences Building Rm 004
>Stony Brook, NY 11794
>On 11/14/06,  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.
>>Assistant Professor
>>Department of Biology
>>University of Central Oklahoma
>>100 N. University Drive
>>Edmond, OK 73034

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