[HISTONET] embedding cell cultures

[Sherwood, Margaret]

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   

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Geoff McAuliffe
Sent: Wednesday, January 27, 2010 3:20 PM
To: Nicholas David Evans
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: Re: [HISTONET] embedding cell cultures

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

Nicholas David Evans wrote:
> 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
>
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>
>   


-- 
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**********************************************
Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583 
mcauliff <@t> umdnj.edu
**********************************************



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