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

[MVaughan4 <@t> ucok.edu]

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
http://www.biology.ucok.edu/PersonalPages/mvaughan/Default.htm

Message: 6
Date: Mon, 13 Nov 2006 12:45:12 -0700
From: Gayle Callis <gcallis <@t> montana.edu>
Subject: Re: [Histonet] Cells Embedded in Collagen for Frozen Tissue
                 Sectioning
To: "Omer Richman" <orichman <@t> gmail.com>,
                 Histonet <@t> lists.utsouthwestern.edu
Message-ID:
 <6.0.0.22.1.20061113123703.01b3b698 <@t> gemini.msu.montana.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed

You do not need a collagen matrix to make a frozen cell block.
Take 3 x 10 to the 7th ( higher power 7) cells suspended in PBS without 
any 
protein carrier.  Spin down and wash 3 times with PBS, after the last 
spin, 
add approx 1 ml or less of OCT to the cell pellet, resuspend the cells. 
Snap freeze end of tube in liquid nitrogen and using a sharp rap on bottom 

of tube, dislodge the block, mount on a chuck and do frozen sections.  If 
you end up with too many cells packed together, cut a thinner frozen 
section.  If the end of the block seems a bit bare or you need to build up 

block, merely add more OCT around the block. The end of the block is where 

the cells are going to be as long as you don't add an enormous amount of 
OCT before snap freezing.  Chris van der Loos also does this and can 
cryosection the most amazingly  tiny block!!

We have had zero luck using a collagen matrix to capture cells and went to 

the above method with far greater success. This is also a nice way to make 

positive controls for immunostaining or Beta Gal methods.

Good luck


Take At 12:19 PM 11/13/2006, you wrote:
>Dear Histonet,
>
>My PI has been embedding cancer cells in collagen over the past several
>weeks. My job is to take the collagen bead (approx 1-1.5 cm in diameter),
>embed it in OCT and get frozen sections for H&E staining. Unfortunately, 
I
>have been having a very hard time finding the cells in the collagen in 
the
>OCT block because there is little to no contrast between the two. On top 
of
>the that, the collagen detaches itself from the OCT as it hits the blade
>leaving me with a square section of OCT that is missing a circle in the
>middle (presumably where the collagen and cells would be if I could see
>them).
>
>I have gone through several full specimens and have come away with only a
>slide or two where I managed to catch some cells. This sort of 
inconsistency
>is obviously not acceptable and my resources in the pathology department
>have come up dry. Does anyone have any experience with this sort of
>experiment? Is there any way to stain the collagen/cells in such a way 
that
>I could at least tell where the cells are in the OCT block so I'm not 
just
>blindly cutting away? Any help would be greatly appreciated. Thanks to
>everyone in advance.
>
>Sincerely,
>
>Omer Richman
>Research Technician
>State University of New York at Stony Brook
>Life Sciences Building Rm 004
>Stony Brook, NY 11794
>_______________________________________________
>Histonet mailing list
>Histonet <@t> lists.utsouthwestern.edu
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet

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
406 994-4303 (FAX)