[Histonet] Cells Embedded in Collagen for Frozen Tissue Sectioning

[Gayle Callis]

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
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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
406 994-4303 (FAX)