Re: DiD / DiI / DiO versions 'solid' and 'oil'

Posted by Steffen Dietzel on
URL: http://confocal-microscopy-list.275.s1.nabble.com/sorry-tp593651p593655.html

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Hi Mike,

thanks for your response. Since this is going to be pretty specialized, I reply off list. First, I also asked a similar question via [hidden email], so the orignal question may end up in your inbox twice.

Second, the tracer sampler kit would be nice if it weren't for the Ca-detectors I also have to use. So I have to stay clear of the green and preferably orange channels, that's why I am looking (only) into DiD in the first place.

With the pastes: We want to put the dye into intact blood vessels, diameter maybe 100-200 µm. I don't think we are going to manage to stuff the paste inside. What do you think?

We can let solutions flow through it via glass capilaries that are attached to the ends, so I planed to let some buffered solution flow in, incubate for let's say 30 min and then wash again with buffer before fluorescence microscopic observation. Does that sound like a doable experiment for you, regarding the DiD staining?

Since I didn't find much details about the differences of the oil and solid versions, I brought it to the confocal list.

As a side note, the DiD paste ( N22882) dosn't seem to have an entry on the web site and thus no price information.


thanks for your help in this

Steffen


At 20:28 16.07.2008, you wrote:

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I concur Sylvie response - I also would be concerned that any holes in the endothial layer would allow oil version to leak out.  Better containment with the paste - applied with glass needles.

To make protocol optimization a tad easier, we offer two sampler kits (below).  For details our web site is best, rather than blogging here.

Lipophilic Tracer Sampler Kit  The Lipophilic Tracer Sampler kit contains 1 mg samples of nine different membrane stains.  SKU# L-7781  This has more than just the DiI versions in case they work out better.

But for just paste versions:  (Ignore neuro - can work for any cell type.)
NeuroTrace® Multicolor Tissue-Labeling Kit - DiO, DiI, DiD pastes, 500 mg each  SKU# N-22884

Mike Ignatius

Molecular Probes/Invitrogen


-----Original Message-----
From: Confocal Microscopy List [[hidden email]] On Behalf Of Sylvie Le Guyader
Sent: Wednesday, July 16, 2008 1:01 AM
To: [hidden email]
Subject: Re: DiD / DiI / DiO versions 'solid' and 'oil'

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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Steffen

From my work in Zebrafish I'd recommend to purchase DiD solid so you can
dissolve it in what you want. It might be difficult to get it to mix well in
blood if it is diluted in oil.

In live fish we were using DiI and DiO diluted about 5% in Ethanol. You'll
definitely get some dye leaks to neighbouring cells after a few hours so you
will need to image fairly quickly. For fixed fish we were using a saturated
solution of DiI in chloroform. Leaks also occur but it takes several days at
4º.

If you get the solid form, you can try a few things and see what works best.
Powders are kept in a dry box.

Med vänlig hälsning / Best regards
 
Sylvie
 
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader
Dept of Biosciences and Nutrition
Karolinska Institutet
Novum
14157 Huddinge
Sweden
[phone]


> -----Original Message-----
> From: Confocal Microscopy List [[hidden email]]
> On Behalf Of Steffen Dietzel
> Sent: 15 July 2008 19:13
> To: [hidden email]
> Subject: DiD / DiI / DiO versions 'solid' and 'oil'
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hello there,
>
> the DiI / DiO related membrane stain DiD from
> Molecular Probes comes in two flavors (as do its relatives):
>
> 'DiD' oil = DiIC18(5) oil (cat# D307) or as 'DiD'
> solid = DiIC18(5) solid (cat# D7757).
>
> Can anyone comment on what the
> advantages/disadvantages of the two flavors are?
>
> The idea for the experiment is to use DiD to
> stain the membranes of endothelial cells of blood
> vessels (from the interior of the vessel), to be
> able to distinguish the endothelial cells better
> from neighboring smooth muscle cells (SMCs)
> Povided that the endothelium is intact, the dye
> should not be able to reach the SMCs - or so ist
> the hope. DiD should be sufficiently in the red
> to allow the simultaneous usage of the green
> channel e.g. for Fluo-4 Ca measurements.
>
> Regards
>
> Steffen
>
> --
>
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> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
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