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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]>
>>
>
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