Re: cell monolayers and aclar

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In a message dated 05/02/2000 6:32:35 AM US Mountain Standard Time, writes:

<< Has anyone used ACLAR (Ted Pella) to grow cell monlayers on (like 
 coverslips) and then processed them for EM. Are there any other ways to do 
 this ....I have just started looking into it. Also, if anyone has a protocol 
 for H&E staining of Lowicryl I would appreciate borrowing it?
 Thank you, as usuall:


I used to do exactly this kind of thing with Aclar, and it worked pretty 
well.  We cultured the cells on small rectangles of Aclar, with one corner 
cut off so we could tell quickly which side the cells were on (for example, 
make the rectangles about 2 mm wide and 4mm tall, then cut off the upper 
right-hand corner).

We hand-processed the rectangles and embedded in Spurr low viscosity resin in 
flat silicone embedding molds, with the cells facing up (into the main mass 
of the block).  The Aclar will sink to the bottom of the mold.

After polymerization you can strip the Aclar out of the block.  This will 
leave a small depression in the block where the Aclar was located, but the 
monolayer will stay in the mass of the block.  

Re-orient in a flat mold with the depression facing up.  Add a few drops of 
fresh Spurr to fill the depression and polymerize once again.  You will then 
have one solid block with the cultured cells nicely sandwiched in the Spurr 
resin.  Mount and trim as usual, so that you cut the cells in cross-section 
as a monolayer.

I didn't have many problems with the Spurr resin splitting between the 
layers.  Most of the blocks cut very nicely.

Good luck, let us know how it turns out!


Bob Chiovetti
GTI Microsystems
Tucson, AZ

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