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Dear Kate,
First, try cutting off the feet from the insert with a razor blade.  If
you want ensure medium exchange during preliminary culture then rest
the insert on glass capilliaries, pins, etc.  Turning the insert over
and culturing on the 'bottom' surface will work, our slices tend to
stick to the membranes unless dislodged by gentle prodding with a
spatula or a stream of medium from a pipet.  Hypoxia issues will depend
upon oxygenation of your medium and section thickness - if thick enough
then you can trim the membrane feet to provide some spacing for flow
around the slice.

Regards,
Glen

Search the CONFOCAL archive at
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> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hello,
> I culture brain slices on millipore inserts. I plan to do some real
> time
> imaging on a confocal (for 24-48hrs). My problem lies with the insert.
> The
> confocal Im using has the objectives underneath the sample which means
> that
> it has to image through the insert membrane. We have found that the
> microscope can not do this. The inserts have small 3mm feet and a top
> height of 5mm. I could try inverting the whole insert but I think the
> media
> would then have to go above the insert which may cause the slice to
> fall
> off, also it would probably make a seal between the insert and the dish
> making my slices hypoxic. Somebody has given me some matrex dishes
> which
> have a coverslip bottom and ideally I need to place my slices on them
> and
> cover them with media. Im not sure if one side of the slice needs to be
> exposed to facilliate gas exchange - any comments on this and inserts
> would
> also be appreciated, has anyone tried it before?.
> Kate.x.

Kate
Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
(206) 616-4156
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