In message  <[log in to unmask]> Confocal Microscopy List writes:
> Dear Confocalists,
> I am trying to determine the shapes of the cells grown in dense 3-D
> cultures by means of membrane staining dye DII. The problem is that I
> have to fix the cells before the observations. Both formaldehyde and
> glutaraldehyde that I used for fixation seem to destroy cell
> membranes so their fluorescence after staining is barely observable. I
> would greatly appreciate any sugestions concerning fixation protocols
> or solutions that would fix the cells not destroying the structure of
> their membranes.
> Thanks in advance,
>
> Jarek Czyz
>
> Dr. Jaroslaw Czyz
> Department of Cell Biology
> The Jan Zurzycki Institute of Molecular Biology
> al. Mickiewicza 3
> 31-120 Krakow
> Poland
> [log in to unmask]

Hi I have never used DiI myself, but as I understand it the problem may not be
destruction of the membranes by fixative (this should not happen with
glutaraldehyde), but just that the DiI is not fixed and therefore subsequently
leaches out of your preparation before you get to view it (Of course, you must
not use any detergent or solvent steps in your procedure!). Molecular probes
sell a number of DiI derivatives which are reported to be more fixable. The one
which looks especially good (at least from the catalogue) is SP-DiIC18 (D-7777).
This shows almost 100% retention after formaldehyde followed by acetone, whereas
the normal diI staining is reduced by over 60% due to the acetone.

It may be worth giving it a try if it isnt too expensive!

Matthew


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     Dr. Matthew J Hannah
     Institute for Neurobiology
     University of Heidelberg
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