Re: silicone membrane sections
Other folks with more plastic embedding experience will probably
write you, so I'll stick to something else that I hope will be
Fix and dehydrate the cells using a TEM routine (Karnovsky's fixative
+ 1% tannic acid, gentle dehydration, etc.) Once in absolute ethanol,
plunge-freeze the cells + discs into LN2^.
Note: since the cells are dehydrated, and ethanol freezes as a glass
(without any crystal structure), then ice damage is unlikely or will
Once frozen, snap the discs in two or more pieces *while in ethanol
in LN2* and then critical-point dry the discs, or dry using
hexamethyldisilizane. See http://www.biotech.ufl.edu/EM/ for more
Examine the broken edges of the discs using a scanning EM. The pores,
and any cellular penetration into the pore space, will be directly
seen. Take stereopair images. Judging from what I've seen before,
I'd be willing to bet there will be little penetration.
The SEM should be available at the University of Sheffield.
^Cut a piece of Parafilm, fold in 2, then fold in and crimp 3 edges,
to make a sealed pocket. Fill with absolute ethanol, place one sample
inside, crimp the remaining edge to seal. Throw the whole package
into the LN2.
>I have had a request to cut sections from a novel porous silicone
>discs onto which is grown ciliated epithelium.
>The silicone membrane is made buy mixing a silicone plastic with fine
>salt grains and extruding the mixture through a circular dye to form
>These cylinders are cut into circular discs.
>The discs are washed in water to remove the salt which leaves an open
>mesh network of the plastic.
>Fibroblasts are grown on the discs and then ciliated epithelium is
>grown on the fibroblasts.
>The object is to cut sections to see the penetration of cells into
>the open structure of the disc.
>I have tried processing into wax but the plastic is too soft and it
>pulls away from the wax.
>Soaking the disc in gelatine and fixing before processing does not
>help either because the plastic remains flexible.
>I have tried frozen sections but again the plastic remains flexible
>at -20C and will not section.When looking at the sections down the
>microscope is is clear that the plastic is pulling away from the wax.
>Freezing in liquid nitrogen makes the discs stiff but we don't have a
>cryostat which will get down to those temperatures.
>Does anyone know of a way to harden the plastic without artifactually
>damaging the cells grown upon it?
>I would love to here from workers who have solved this problem.
>Sheffield Children's Hospital UK
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Supervisor, BBPIC microscopy facility
Department of Animal Sciences
University of Wisconsin
1675 Observatory Drive
Madison, WI 53706 - 1284
voice: (608) 263-4162
fax: (608) 262-5157 (dept. fax)
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