[Histonet] Cell Pellets in Agar

[Helen Fedor]

Paul, Here is the protocol that we use in our Lab.
Helen

Helen L. Fedor B.S.
Johns Hopkins University
Pathology Department
600 N Wolfe St
Marburg Room 406
Baltimore MD 21287
email: hfedor <@t> jhmi.edu
Phone: 410 614-1660
Pager: 410 283-3419

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>>> "Monfils, Paul" <PMonfils <@t> Lifespan.org> 01/16/06 10:31 AM >>>
	You can use a number of soluble, coagulable proteins to bind cell
pellets together, including agar, agarose, gelatin, albumin, serum, etc.  I
use Mayer's albumin, but the technique is pretty much the same regardless of
which substance is used.  After fixing the cells in suspension, I spin them
down and remove the fixative.  Then wash a couple of times in buffer by
resuspending and spinning down. After the last wash I resuspend in buffer
once more, add 2 drops of Mayer's albumin per ml of suspension, mix
thoroughly by vortexing a few seconds, allow to stand for a couple of
minutes, then spin down once more and remove the supernatant.  I then begin
dehydration of the pellet in the centrifuge tube, usually beginning with 50%
ethanol, then 70%, 95%, and absolute ethanol. Pipette the initial alcohol
gently down the side of the tube to ensure that the pellet is not disturbed.
Once you are in the stronger alcohols the thin coating of albumin on the
cells has coagulated, binding them firmly together.  At some point in the
dehydration series the pellet, due to contraction during dehydration,
usually detaches from the tube intact. If it doesn't actually detach, it can
be detached either by drawing up alcohol into a pipette and squirting it
onto the pellet, or if necessary, by the slightest touch of a slender probe.

	I then either wrap the pellet in lens paper and place it in a
cassette on the tissue processor, starting with the final absolute alcohol
station, or, I finish the processing by hand, in the centrifuge tube.  If
you are going to do the latter, make sure your tube is resistant to the
clearing agent.  Polypropylene tubes work best for me.  Polystyrene tubes
are more transparent, and are fine if you are only going to use alcohol in
the tube, but many clearing agents dissolve polystyrene.



> ----------
> From: 	histonet-bounces <@t> lists.utsouthwestern.edu on behalf of
> Johns, Laura [CNTUS]
> Sent: 	Monday, January 16, 2006 5:59 AM
> To: 	'histonet <@t> lists.utsouthwestern.edu' 
> Subject: 	[Histonet] Cell Pellets in Agar
> 
> Does anyone have a protocol for paraffin embedding cell pellets?  We've
> had
> problems with cell pellets staying together after fixation and during
> embedding, and I know that occasionally people use agar or agarose to keep
> the pellets compact.  Is it better to use agar or agarose? 
> 
> Thanks,
> 
> Laura M. Johns, Ph.D.
> Centocor, Inc.
> 145 King of Prussia Road (Mail Stop # R-4-2)
> Radnor, PA 19087
> Phone:  610-240-8284 ....  Fax:  610-240-8150....  Email:
> ljohns53 <@t> cntus.jnj.com 
> 
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