Re: DiD / DiI / DiO versions 'solid' and 'oil'
Posted by
George McNamara on
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Hi Steffen,
Check out Ravnic et al 2005 Microvasc Res 70: 90, Pubmed
16095629:
"The fluorescent dye
1,1-Dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate was
obtained from Sigma (St. Louis, MO). The carbocyanine dye was dissolved
in
ethanol (6 mg/ml) and stored as a 6.42 mM stock solution at
4-
C. Immediately prior to infusion, the stock
solution was diluted in phosphate buffered saline (PBS) containing
glucose (200 mM) for a final concentration of 0.128 mM."
Was used to label mouse vessels. The inventor of the technique,
Rong Wen, has a paper submitted to Nature Protocols, so please keep an
eye out for it (shows the entire endothelial cell takes up the dye and
has a terrific image of a sprouting cell). Rong told me that diluting the
stock immediately before is critical. Glucose is better than no glucose.
Rong usually asphyxiates (spelling?) the mouse with CO2, but a smaller
amount of DiI can be injected for live mouse imaging, in which case it
mostly coats RBCs. Rong like the Sigma-Aldrich dye (less pure) because it
is less expensive ($0.50/mouse) than Invitrogen's. I have not checked
what other DiFamily members Sigma-Aldrich has. I also have not calculated
how expensive the 1:1 Alexa Fluor 647-streptavidin : biotin-tomato lectin
mix used in my upcoming Paddock 2.0 book chapter is, but Rong's DiI is a
lot less expensive.
Brant Weinstein used to inject zebrafish blood vessels with dyes, though
he has mostly switched to fluorescent proteins, as in:
Kamei M, Weinstein BM. Long-term time-lapse fluorescence imaging of
developing zebrafish. Zebrafish. 2005;2(2):113-23. PMID:
18248171.
See also his web site at NIH.
Best wishes,
George
At 04:17 AM 7/17/2008, you wrote:
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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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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
+46 (0)8 608 9269
> -----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
>
> --
>
----------------------------------------------------------------------------
-----------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
>
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George McNamara, Ph.D.
University of Miami, Miller School of Medicine
Image Core
Miami, FL 33010
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