Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi Winnok,
I guess I'd try hexidium iodide first - it's (relatively) cheap, and you can
always continue to use it as a nuclear counterstain like PI if it doesn't
get into your cells or is too toxic even at low concentration. We use HI at
10 micrograms/ml or less, just in water or buffer.
I don't know about animal cells, but in our hands, unless you have the
concentration exactly right and time your observation quite carefully, the
FM dyes will get into a number of membranes, they're not very specific. And
concentration and time are different for different tissues. The Syto dyes
are even worse that way. That's the beauty of PI and HI, they seem
reasonably specific for DNA (and plant cell walls, of course).
Re. efficiency of 488 nm excitation, it depends how good your lasers are.
The 543 HeNe laser on our Leica SP2 is a wimp, and the 488 line of the Ar
laser does a perfectly fine job for PI excitation.
cheers,
Rosemary
> From: winnok <[log in to unmask]>
> Reply-To: Confocal Microscopy List <[log in to unmask]>
> Date: Wed, 14 Sep 2005 09:44:22 +0200
> To: [log in to unmask]
> 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
>
> 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]>
>>>
>>
>
|
