SPAM-LOW: [Histonet] Re: Histonet Digest, Vol 74, Issue 34

[Patsy Ruegg]

What I do is make a cell block.  If you can get the spheroids into
suspension in a test tube, you could fix in formalin, spin them down in a
centrifuge and then take of the supernatant, add warmed histogel and
resuspend in the histogel, spin it down to get your spheroid in the bottom
of the tube, then let the histogel cool so it will harden, you can then
tease the hardened plug out of the tube, wrap it in paper and process into
paraffin, keep track of the bottom where your cells should be and embed them
down, they should be on the surface of your paraffin tissue block for
sectioning.  

Another way is if you can see the spheroids, after fixing, move them with
fine forceps, you could fill an embedding mold with warm liquid histogel,
then place the spheroid in the mold, cool the histogel til it hardens, then
pop it out of the mold and wrap in paper and process into paraffin as the
cell block above.  We get HISTOGEL from Richard Allan. 

Regards,

Patsy 

Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net 
www.ihcrg.org


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Rhonda
Henshall-Powell
Sent: Thursday, January 28, 2010 12:09 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: SPAM-LOW: [Histonet] Re: Histonet Digest, Vol 74, Issue 34

Dear Nick,

When I was growing and harvesting 3D cell cultures (spheroids grown in
matrigel) we would remove the culture medium, draw up the gel in a
needle-less syringe and place in to OCT embedding medium (Tissue Tek) before
freezing in Liquid Nitrogen.  I would then cut 5-10um sections and perform
IHC or immunofluorescence.

Hope this helps - but it looks like you have a lot of good suggestions
already.

Best Regards,
Rhonda

Rhonda Henshall-Powell, Ph.D.

> 
> ------------------------------
> 
> Message: 2
> Date: Wed, 27 Jan 2010 10:25:42 -0800 (PST)
> From: Nicholas David Evans <ndevans <@t> stanford.edu>
> 
> Dear all,
> 
> I was hoping someone might be able to offer me some advice
> on embedding and sectioning cell cultures.
> 
> In short we are growing cells which form 3D dome-like
> structures on tissue culture plastic. Does anyone have any
> experience or advice to offer on embedding the cultures in
> situ before sectioning? I have seen various methods in the
> literature, which often use Epon to embed the material
> followed by sawing away the plastic, but if anyone can offer
> some tips on other possible (easier) ways of doing it, or
> can refer me to some useful literature, I'd be very
> grateful.
> 
> I would like to have simple 10um sections at 90 degrees to
> the substrate, which I can use for IHC.
> 
> Best wishes
> Nick
> 
> 
> 
> ------------------------------
> 
> Message: 3
> Date: Wed, 27 Jan 2010 15:19:36 -0500
> From: Geoff McAuliffe <mcauliff <@t> umdnj.edu>
> 
> Hi Nick:
> 
> You can use the Epon substitutes such as EmBed 812. Fix,
> osmicate, 
> dehydrate as usual, but omit the proplylene oxide as it
> will react with 
> the plastic dish. Epon substitutes will mix with ethanol. I
> used 2:1 
> then 1:1 then 1:2 ratios of absolute ethanol to "Epon" with
> catalyst 
> added with agitation. Then several changes of pure Epon and
> polymerize. 
> Yes, you will have to saw away the plastic, if you try to
> section the 
> Epon-plastic combo the two will separate.
> How much easier do you want it to be?
> 
> Geoff
> ------------------------------
> 
> Message: 5
> Date: Wed, 27 Jan 2010 15:39:05 -0500
> From: "Sherwood, Margaret " <MSHERWOOD <@t> PARTNERS.ORG>
> Subject: RE: [HISTONET] embedding cell cultures

> Nick,
> 
> I am assuming that your 3D cells only grow on
> plastic.  They make plastic cover
> slips which, if your cells attach to them and grow, might
> make the embedding
> much easier.  You would follow the same method as
> stated, but then you could
> invert the coverslips on a beem capsule and separate the
> coverslip from capsule
> with liquid nitrogen.  However, I have never done it
> with plastic coverslips
> (only glass), so not sure if they would easily separate
> from capsule with liquid
> nitrogen. If anyone else has done so, please weigh in. 
> 
> Peggy   
> 
> ------------------------------
> Message: 6
> Date: Wed, 27 Jan 2010 15:44:37 -0500
> From: Peggy Bisher <mbisher <@t> Princeton.EDU>
> Subject: Re: [HISTONET] embedding cell cultures
> 
> I have done just what you are talking about using Aclar. It
> is a plastic
> embedding film (purchased from EMS). It works great for
> us.
> 
> Margaret E. Bisher
> Electron Microscopy & Histology Core Facility Manager
> Department of Molecular Biology
> Princeton University
> Moffett Laboratory, Room 113
> Princeton, New Jersey
> Office: (609) 258-7026
> Fax: (609) 258-8468
> mbisher <@t> princeton.edu


      

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