Tissue prep question

[Mike Tighe]

I have been trying to cut thick sections (50-200um) of fresh spleen and Lymph node on a vibrating microtome. I have tried to embed in low melt agarose but I have been unable to get uniform sections. What seems to happen is that the knife pushes the tissue out of the way instead of cutting and eventually tears large chunks of tissue all at once. Does anybody have any suggestions?

Thanks for any help!
Mike

[Martin Wessendorf-2]

Dear Mike--

Mike Tighe wrote:
 > I have been trying to cut thick sections (50-200um) of fresh spleen
and Lymph node on a vibrating microtome. I have tried to embed in low
melt agarose but I have been unable to get uniform sections. What seems
to happen is that the knife pushes the tissue out of the way instead of
cutting and eventually tears large chunks of tissue all at once. Does
anybody have any suggestions?

--Make an "L"-shaped chuck out of plastic.  The upright of the "L" will
be perpendicular to the plane of section and sit behind the tissue; the
bottom of the "L" will be parallel to the plane of section and sit below
the tissue.  Place a chunk of agar (--the cheap stuff is fine) between
the upright of the "L" and your tissue.  That will stabilize your tissue
while cutting and won't destroy your blade when your section cuts free.

Credit for this idea goes to the lab of John Williams out at OHSU.

Good luck!

Martin Wessendorf

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Rosemary.White]

Dear Mike,
One thing we invested in was a sapphire knife - made all the difference when
sectioning tricky plant tissues, though spleen probably isn't tricky.  These
knives last a long time, too.
cheers,
Rosemary

Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

T 61 2 6246 5475
F 61 2 6246 5334
M 61 2 420 972 028
E [hidden email]



On 14/04/10 7:22 AM, "Martin Wessendorf" <[hidden email]> wrote:

> Dear Mike--
>
> Mike Tighe wrote:
>> I have been trying to cut thick sections (50-200um) of fresh spleen
> and Lymph node on a vibrating microtome. I have tried to embed in low
> melt agarose but I have been unable to get uniform sections. What seems
> to happen is that the knife pushes the tissue out of the way instead of
> cutting and eventually tears large chunks of tissue all at once. Does
> anybody have any suggestions?
>
> --Make an "L"-shaped chuck out of plastic.  The upright of the "L" will
> be perpendicular to the plane of section and sit behind the tissue; the
> bottom of the "L" will be parallel to the plane of section and sit below
> the tissue.  Place a chunk of agar (--the cheap stuff is fine) between
> the upright of the "L" and your tissue.  That will stabilize your tissue
> while cutting and won't destroy your blade when your section cuts free.
>
> Credit for this idea goes to the lab of John Williams out at OHSU.
>
> Good luck!
>
> Martin Wessendorf