Jarek Czyz wrote:

>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.

As someone who *has* used DiI a great deal, I can say unequivocally that
formaldehyde fixation does not prevent DiI labelling. In fact, the great
advantage of the lipophilic dyes is that they can be used in fixed tissue.
I haven't ever
tried gluteraldehyde, my guess is that it would be fine for DiI labelling but
give more autofluorescence.

That said, perhaps there is something else wrong with your protocol? As
someone already mentioned, use of detergents or other membrane-extracting
agents would of course be a problem! Also, how are you applying the DiI to
these cultures? It is most often used as a neuronal tracer for anterograde
or retrograde labelling, rather than for mass labelling of a culture. It
should in principle be possible to label all of the cells in a culture, but
you need some way of delivering the highly hydrophobic dye through the
culture medium to reach the cell membranes. The protocol that I know of was
developed by Steve Potter in Jerry Pine's lab at Caltech, and uses the
nonionic detergent Pluronic F-127.

Good luck,
Chi-Bin Chien




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