Re: cell invasion on gels

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From:Jeffrey S Crews <> (by way of histonet)
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My earlier message was not sufficiently clear. Sorry 'bout that. I was
thinking of our usual application, which is collagen cast on a
semipermeable membrane, and contracted with fibroblasts so that it is
robust and easy to handle. If you have looser, "snotty" collagen gels and
are growing them on plates, this is how we've handled those:
	Fix gel on the plate to make it firmer. Then scrape off the largest
sheet you can with a razor. Flood with PBS and float the sheet out of the
culture plate and into another dish of PBS in which you have placed a
piece of lens paper or Surgipath Biowraps. Arrange the collagen back into
a nice flat unwrinkled sheet (easy to do while it's submerged) then pipet
out the PBS, leaving the collagen stranded on the lens paper. Cover with
another piece of lens paper.  Handle this paper-collagen sandwich as one
piece for all subsequent steps. Cut out a piece of sandwich that will fit
in a cassette, process, then trim to about 3mm wide. Embed on edge.
	Cut and stain. The lens paper does not adversely affect cutting, and
does not stain with H&E. The uncolored line of cellulose fibers above and
below the sample are easy to distinguish and won't alarm anyone as long
as you tell them what they are. Collagen gels stain a very light, almost
white pink with eosin and the fibroblasts are easy to distinguish.
	This technique works really well and allows you to see cross
sections of
gels that you would think were impossible to handle. Remember that when
they dehydrate, the samples will become radically thinner due to their
high water content.

On Fri, 7 Jan 2000 11:16:06 -0700 Gayle Callis
<> writes:
>We did this but had to make sure gels were embedded on edge, not an
>easy thing to do, and keep it flat.  Our best success was to fix,
>and embed in GMA, then use the skinny embedding molds to make sure
>gel was on edge.  One could also embed, then turn the block on edge,
>and embed again with the gel on edge  This actually
>proved to be the easier technic, to embed gel in GMA, then do the
>double embedding.      It was important to fix the gel while it was on
>plate, or it fell apart if one tried to lift it out unfixed.  The
>could be cut out with scalpel after fixation.  Still dicey!
>The embedded blocks were carefully examined with a hand lens before
>cutting to make sure the gel with cells was in the
>proper orientation, adjust block and section. We were able to detect
>cells growing into the gel fairly easily, using NBF fixation,
>H&E staining.  We experienced better resolution of cells in GMA than
>with paraffin.  No IHC was done, however.
>Gayle Callis

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