Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Winnok,
We have successfully used Draq5 (www.biostatus.co.uk) to label live cell
nuclei. Optimal excitation is 647 so it is in fact far red rather than red,
but is certainly worth considering.
Best,
Stuart Shand
>From: winnok <[log in to unmask]>
>Reply-To: Confocal Microscopy List <[log in to unmask]>
>To: [log in to unmask]
>Subject: Red fluorescent Counterstain for live imaging (was: DiI labeling)
>Date: Tue, 13 Sep 2005 16:37:30 +0200
>
>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]>
>>
|
