Dear Stan,

Here is our protocol for the cell-agarose gel:

1. Make 6% agarose gel (we use type VII low gelling temperature agarose)
and maintain at 37% until use
2. Spin down cells from culture and remove supernatant
3. resuspend pelet in media or PBS (you can do a cell count at this point
to see how many cells you have)
4. carefully (make sure you do not introduce air bubbles) mix equal
amounts of the cell suspension and the aragose (end up with an
agarose concentraiton of 3%).
5. put cell-agarose suspension in small mould and place at 4 deg C for 1/2
hour to let gel.
6. remove gel and snap freeze in OCT.

We have also use 2% agarose and gelatin for this.

For smaller cell numbers we have also resuspended in small amounts of OCT
and snap frozen in very small moulds and then embedded the small cell-OCT
block in OCT.

They all worked pretty well for what we wanted.

I hope this is of help
Best regards
Yak-Nam




> Dear Yak-Nam
> I have just seen your post stating you have used 2% agarose cell blocks in OCT for frozen sections.
> Do you have a specific protocol for this ?
> Regards
> Stan
>
> Stan Stylli
> Department of Surgery / Neurosurgery
> Royal Melbourne Hospital
> Parkville Australia 3052
> Tel : 61-3-93427616
> Fax : 61-3-93477695
>
>
>
> -----Original Message-----
> From: Y. Wang [mailto:ynwang@u.washington.edu]
> Sent: Thursday, 19 August 2004 9:49 AM
> To: Elizabeth Chlipala
> Cc: histonet@lists.utsouthwestern.edu; 'LINDA MARGRAF'
> Subject: RE: [Histonet] fibrin for cell blocks
>
>
> Linda,
>
> We have also used a 2% agarose solution and have been sucessful embedding
> the agarose-cell mixture in OCT for frozen sections.
>
> Yak-Nam
>
> Senior Fellow
> Department of Bioengineering
> University of Washington
> Box 357962
> Seattle, WA 98195
>
> Tel.: (206)-221-5873
> Fax.: (206)-221-5874
>
> On Wed, 18 Aug 2004, Elizabeth Chlipala wrote:
>
> > Linda
> >
> > I use a 2% agarose solution.  Basically, you spin the cells down, remove
> > the supernatant and then re-suspend in the agarose and spin again.  You
> > can remove the agarose pellet, process and embed in paraffin.  I'll send
> > you the pdf of the reference I have used.
> >
> > Liz
> >
> > Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
> > Premier Histology Laboratory, LLC
> > P.O. Box 18592
> > Boulder, Colorado 80308
> > Office: (303) 735-5001
> > Fax: (303) 735-3540
> > lizchlipala@premierhistology.com
> > www.premierhistology.com
> >
> > Ship to Address:
> > Premier Histology Laboratory
> > University of Colorado
> > MCBD, Room A3B40
> > Boulder, Colorado 80309
> >
> > _________________________________
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> >
> > -----Original Message-----
> > From: histonet-bounces@lists.utsouthwestern.edu
> > [mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of LINDA
> > MARGRAF
> > Sent: Wednesday, August 18, 2004 1:29 PM
> > To: histonet@lists.utsouthwestern.edu
> > Subject: [Histonet] fibrin for cell blocks
> >
> > Dear Histonetters:
> > Does anyone have experience using fibrin or another substance as a
> > matrix to add a cell suspension to when  making cell blocks.  My
> > colleague wants to make cell blocks to section and stain for a research
> > study but there will only be a small amount of cellular material.  She
> > remembers seeing this done in the past but needs to know where to obtain
> > the reagnets and what procedures to follow. Thanks so much
> > Linda M
> > Histonet administrator
> >
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