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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Jens,
The qdot655 is visable by eye. We have worked quite a lot with Qdots as
secondary Abs and the 655 is actually the most pleasing to the eye, a real
deep red.
Best
Stu
>From: Jens Rietdorf <[log in to unmask]>
>Reply-To: Confocal Microscopy List <[log in to unmask]>
>To: [log in to unmask]
>Subject: Re: Red fluorescent Counterstain for live imaging (was: DiI
>labeling)
>Date: Wed, 14 Sep 2005 10:49:32 +0200
>
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Dear Winnok et al.,
>
>to be added to the list: we have recently used WGA(lectin)-qdots655 instead
>of
>FM dyes.
>The advantages: (1) Can be excited by several laserlines, (2) do not enter
>the
>cells (which is typically the big disadvantage of qdots, here it is a big
>plus
>compared to FM4-64), (3) bright signal, excellent spectral separability
>from
>all the FPs.
>The disadvantages: (1) expensive, (2) qdot655 not visible by eye (655 was
>the
>only commercially available variant), (3) little experience with side
>effects,
>staining was sometimes a bit uneven or dotty, we havent worked out a good
>protocol yet (if somebody has, I would be extremely intereted).
>
>Cheers, jens
>
>Quoting winnok <[log in to unmask]>:
>
>>Search the CONFOCAL archive at
>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>>Thank you Rosemary for the very complete listing of products.
>>Now I have a choice between
>>
>>- FM 4-64
>>- FM 5-95
>>- Syto Red
>>- Hexidium iodide
>>
>>Which one would give the most specific staining?
>>Looking at the negative aspects of Syto dyes you mentioned, I'll
>>leave this one out.
>>In earlier experiments I have tried FM2-10 (~FM 1-43) but
>>unfortunately with no succes. Supposing all FM dyes are labeled in
>>the same way, does anyone have a good protocol for labeling mammalian
>>cells with them? Isn't there always some trafficking of these dyes in
>>the cytoplasm?
>>Does anybody else have experience with one or more of these stains on
>>mammalian cells?
>>
>>(PS: isn't using 488 for PI a little inefficient? I always use 543
>>excitation.)
>>
>>Thanks,
>>
>>Winnok
>>
>>
>>----- Original Message ----- From: "Rosemary White"
>><[log in to unmask]>
>>To: <[log in to unmask]>
>>Sent: Wednesday, September 14, 2005 12:32 AM
>>Subject: Re: Red fluorescent Counterstain for live imaging (was: DiI
>>labeling)
>>
>>
>>Search the CONFOCAL archive at
>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>>Well, this is from a plant person, we use FM 4-64 and FM 5-95 as general
>>plasma membrane labels in living cells, and they do eventually get into
>>most
>>of the membrane systems in the cell. You'd get a red nuclear membrane
>>eventually.
>>
>>The SYTO dyes work quite well in animal tissues, I hear, although none of
>>the vast number of SYTO dyes I've tried works very well in plants - the
>>dyes
>>label too many other cell components apart from the nucleus and/or are
>>toxic.
>>
>>We use hexidium iodide routinely because it gets into more living cell
>>types
>>than DAPI, but it is slightly toxic and has to be used at low
>>concentration
>>if you want to follow the cells for any length of time. It has a very
>>broad excitation range, like the non-permeant propidium iodide (for which
>>we
>>usually just use the 488 line), and a broad emission range as well. Might
>>be worth a try.
>>
>>cheers,
>>Rosemary
>>
>>Dr. Rosemary White [log in to unmask]
>>Microscopy Centre ph. 61-2-6246 5475
>>CSIRO Plant Industry mob. 61-0402 835 973
>>GPO Box 1600 fax. 61-2-6246 5334
>>Canberra, ACT 2601
>>Australia
>>
>>>From: winnok <[log in to unmask]>
>>>Reply-To: Confocal Microscopy List <[log in to unmask]>
>>>Date: Tue, 13 Sep 2005 16:37:30 +0200
>>>To: [log in to unmask]
>>>Subject: Red fluorescent Counterstain for live imaging (was: DiI
>>>labeling)
>>>
>>>Search the CONFOCAL archive at
>>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>
>>>Dear Andrea
>>>
>>>Thank you very much for your reply. This at least saves me from more
>>>succesless attempts.
>>>I must say that then I was misinformed by the invitrogen service.
>>>
>>>Since DiI won't work on living cells I'll repeat the last part of my
>>>former
>>>mail as a new subject:
>>>Does anybody know of a red fluorescent non toxic stain for imaging the
>>>cell
>>>nucleus or nuclear membrane in living cells?
>>>Thanks in advance
>>>
>>>Winnok
>>>
>>>----- Original Message -----
>>>From: "Dr. Andrea J. Elberger" <[log in to unmask]>
>>>To: <[log in to unmask]>
>>>Sent: Tuesday, September 13, 2005 3:47 PM
>>>Subject: Re: DiI labeling
>>>
>>>
>>>>Search the CONFOCAL archive at
>>>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>>
>>>>WINNOK - Your outcome using DiI was due to the fact that you used it in
>>>>live cells.
>>>>
>>>>Even in neurons, when DiI is applied to live cells it is endocytosed and
>>>>transported in the cytoplasm; in the case of neurons, it is transported
>>>>retrogradely to the cell body.
>>>>
>>>>It is only when you use the DiI in aldehyde-fixed tissue that you see it
>>>>as a lipophilic dye that passively diffuses within the lipid bilayer so
>>>>that the entire cell membrane is labeled.
>>>>
>>>>Sorry, but DiI can't give you the results that you want in these
>>>>conditions. You are right to seek another live counterstain.
>>>>
>>>>ANDREA ELBERGER
>>>>
>>>>
>>>>winnok wrote:
>>>>
>>>>>Search the CONFOCAL archive at
>>>>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>>>
>>>>>Dear all,
>>>>>
>>>>>I try to use DiI-solution (molecular probes) as a counterstain for the
>>>>>membranes of endothelial cells but usually it is visible as vesicles
>>>>>probably due to endocytosis.
>>>>>I have tried several protocols, including the one delivered by
>>>>>molecular
>>>>>probes. I also tried to do the labeling at 4°C to prevent endocytosis
>>>>>but
>>>>>without result, still the stain was visible as vesicles. I searched
>>>>>the
>>>>>list but most discussions about DiI concern neurons or blood vessels.
>>>>>Does anybody have experience with labeling endothelial cells with DiI?
>>>>>Or
>>>>>does somebody know of a good non toxic red fluorescent live
>>>>>counterstain
>>>>>for the nuclear membrane or nucleus, better than DiI?
>>>>>Many thanks in advance,
>>>>>
>>>>>
>>>>>Winnok De Vos (ir.)
>>>>>Academic Assistant
>>>>>________________________________________________
>>>>>
>>>>>Faculty of bioscience-engineering, UGent
>>>>>Department molecular biotechnology
>>>>>Coupure links 653 (R224)
>>>>>9000 Gent
>>>>>Belgium
>>>>>
>>>>>tel nr. 09/264.59.71
>>>>>fax nr. 09/264.62.19
>>>>
>>>>
>>>>--
>>>>Dr. Andrea J. Elberger
>>>>Professor, Anatomy and Neurobiology
>>>>Director, Confocal Laser Scanning Microscope Facility
>>>>The University of Tennessee, Memphis
>>>>855 Monroe Avenue
>>>>Memphis, TN 38163 U.S.A.
>>>>tel: 901-448-4101
>>>>FAX: 901-448-7193
>>>><mailto:[log in to unmask]>
>>>>
>>>
>>
>
>
>
>--
>Dr. Jens Rietdorf
>EMBL, Meyerhofstr.1,
>D-69117 Heidelberg,
>Cell Biology/Biophysics Program,
>Advanced Light Microscopy Facility
>Phone ++49(0)6221 387-467 FAX-306
>http://www.embl.de/almf
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