[HISTONET] embedding cell cultures

[Jo Dee Fish]

Hi Nick,
If you can, grow the cells on thermanox cover slips (NUNC).  Then process
them as normal for embedding in Eponate 12.  I use acetonitrile in place of
the propylene oxide to avoid damaging the coverslip.  When doing the final
embedding and polymerization, invert the coverslip over a drop of resin on a
square of ACLAR sheeting.  This forms a "sandwich" of your cell layer.
After polymerization you separte the resin layer from the ACLAR and the
coverslip (very easy!  No sawing!) and "re-embed" one or more layers on
their side in a resin block.  Now, section away.  You will get beautiful
sections of your cells at a nice 90 degree angle.  The best part of doing it
this way is that you can put the embedded cell layer in the light microscope
and select any area of interest before re-embedding and sectioning.
Good luck!
Jo Dee

~~Jo Dee Fish~~
Senior Research Technologist
The J. David Gladstone Institutes
Co-manager Histology and Microscopy Core
 
Telephone: (415) 734-2567
Fax: (415) 355-0824
E-mail: jfish <@t> gladstone.ucsf.edu
 
Mailing address:
The J. David Gladstone Institutes
1650 Owens Street
San Francisco, CA  94158

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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