Membrane labeling with DiI

[kspencer007]

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Chemistry question for all of you...
                We are labeling intact tissue with DiI and DiO. We've tried live, fixed, and fixed with Tx-100 permeabilization, and even overnight incubation with the dye. Labeling of supporting cell membranes is great; very sharp, not diffuse, and very homogeneous distribution throughout the cellular membrane. However, the cells of interest don't pick up the DiI. All cells are equally exposed to the surface of the tissue. What is this telling us about the lipid chemistry of these cells? Can you suggest any pre-treatment that might help this dye gain plasma membrane access on our cells of interest? We have not yet tried saponin...would you recommend this treatment?
                We've also tried CM-DiI to gain access to the inner membrane. But of course, this just gives us diffuse intracellular staining, not membrane-specific.
                I can't find the mechanism of DiI insertion into the plasma membrane; does it prefer a certain class of lipids, that happens to be missing in our cells?
                Thank you for any suggestions.
                Best;
                Kathy

Kathy Spencer
The Scripps Research Institute
Dept of Molecular and Cellular Neuroscience
10550 N. Torrey Pines Road
DNC 210
La Jolla, Ca 92037

[Michael Model]

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These dyes can act strange. We recently tried to stain lipid vesicles with
DiO in the presence of cationic dyes, and DiO staining didn't work in this
combination for no obvious reason. But I also don't quite understand why
you try to stain membranes treated with Triton X100 if it solubilizes
lipids... I suggest, try FM1-43 or something else

Mike Model

On Fri, Oct 23, 2015 at 12:44 PM, Kathryn Spencer <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Chemistry question for all of you...
>                 We are labeling intact tissue with DiI and DiO. We've
> tried live, fixed, and fixed with Tx-100 permeabilization, and even
> overnight incubation with the dye. Labeling of supporting cell membranes is
> great; very sharp, not diffuse, and very homogeneous distribution
> throughout the cellular membrane. However, the cells of interest don't pick
> up the DiI. All cells are equally exposed to the surface of the tissue.
> What is this telling us about the lipid chemistry of these cells? Can you
> suggest any pre-treatment that might help this dye gain plasma membrane
> access on our cells of interest? We have not yet tried saponin...would you
> recommend this treatment?
>                 We've also tried CM-DiI to gain access to the inner
> membrane. But of course, this just gives us diffuse intracellular staining,
> not membrane-specific.
>                 I can't find the mechanism of DiI insertion into the
> plasma membrane; does it prefer a certain class of lipids, that happens to
> be missing in our cells?
>                 Thank you for any suggestions.
>                 Best;
>                 Kathy
>
> Kathy Spencer
> The Scripps Research Institute
> Dept of Molecular and Cellular Neuroscience
> 10550 N. Torrey Pines Road
> DNC 210
> La Jolla, Ca 92037
>

[Alice Schmid]

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No saponin!  If you permeabilization the membranes, you'll lose the DiI.

DiI inserts itself into the space between the lipid bi layers of the
membrane.  If the DiI has access to the membrane, it'll get sucked up.  If
there is a huge ECM that might prohibit access.  So I'd work on figuring
out what is keeping the DiI away from the cell membranes.  But minimal
intervention is the way to go......

Alice Schmid

On Friday, October 23, 2015, Kathryn Spencer <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Chemistry question for all of you...
>                 We are labeling intact tissue with DiI and DiO. We've
> tried live, fixed, and fixed with Tx-100 permeabilization, and even
> overnight incubation with the dye. Labeling of supporting cell membranes is
> great; very sharp, not diffuse, and very homogeneous distribution
> throughout the cellular membrane. However, the cells of interest don't pick
> up the DiI. All cells are equally exposed to the surface of the tissue.
> What is this telling us about the lipid chemistry of these cells? Can you
> suggest any pre-treatment that might help this dye gain plasma membrane
> access on our cells of interest? We have not yet tried saponin...would you
> recommend this treatment?
>                 We've also tried CM-DiI to gain access to the inner
> membrane. But of course, this just gives us diffuse intracellular staining,
> not membrane-specific.
>                 I can't find the mechanism of DiI insertion into the
> plasma membrane; does it prefer a certain class of lipids, that happens to
> be missing in our cells?
>                 Thank you for any suggestions.
>                 Best;
>                 Kathy
>
> Kathy Spencer
> The Scripps Research Institute
> Dept of Molecular and Cellular Neuroscience
> 10550 N. Torrey Pines Road
> DNC 210
> La Jolla, Ca 92037
>