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

From:"Omer Richman"

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:
> Omer,
> 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.
> Mel
> 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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