[Histonet] Re: Histonet Digest, Vol 74, Issue 34

[Rhonda Henshall-Powell]

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