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Gregory, We had the same problem with Alexa 647 and specifically with some 
pollen tubes. Some time, you need to use a 650 LP emission filter to get 
the signal. We tried all switching the antibodies and that sort of things, 
but it did not work. Then we tried directly put a drop of the working 
concentration dye on the slide with a cover slip the signal is nice. You 
can try increase the concentration of the dye to 1:25 or 50 some time to 
get a decent signal. Especially dyes such as Alexa 647 which has a lower 
dye moles (4.2) as opposed to Alexa 488 (around 6.2). Contacted MP and they 
agreed to send another batch but when I asked about the other customers 
they never had problems with the sale of that batch from other customers. I 
do not know what filter you use. For some reason, even while using the 
restricted Em filter (660 LP), the 637 nm red diode laser excites the CY5 
even the signal is really too weak. I know from Jackson Immuno that the dye 
moles are even lower for this compared to Alexa 647, but the probe is 
structurally very different hence, may work with some tissues better. 
However, I have not tried direct comparisons yet in a single tissue under 
comparable concentrations.

No commercial interest with neither of the aforementioned companies.
Shiv

Mayandi Sivaguru, Ph.D.,
Associate Director
Molecular Cytology Core Facility
Molecular Biology Program
2, Tucker Hall
University of Missouri
Columbia, MO 65211-7400
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Voice: 1-573-882-4895
Fax: 1-573-882-0123

www.biotech.missouri.edu/mcc/



At 04:00 PM 11/10/03 +0100, you wrote:
>Search the CONFOCAL archive at 
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>We are having a problem related to one of fluorophores that until now we 
>have not been able to resolve.
>We see and can capture good images when use Alexa488 and Alexa546 labeled 
>probes.
>However, we seem to have a specific problem related to the Alexa633 probe. 
>When use a
>Alexa633-Phalloidin conjugate, which labels f-actin, we see intense 
>staining under the microscope and can
>capture good quality images even when the 633 nm laser wavelength is set 
>to 30%.
>However when we use either an antibody directed against mouse or rabbit 
>labeled
>with Alexa633 (and from two different lots) we obtain at best very weak 
>staining eventhough
>when we use the same primary with the equivalent Alexa546-secondary 
>antibody, we see a very intense signal.
>
>I have obviously contacted Molecular Probes about this issue first and 
>whilst they have provided
>us with a replacement, the problem has not been resolved. Consequently, I 
>was wondering whether
>any of you have any suggestions or have experienced problems with this 
>Alexa633 dye?
>Gregory A. O'Sullivan PhD.
>Dept. of Neurochemistry,
>Max-Planck-Institute für Hirnforschung,
>Deutschordenstrasse 46,
>D-60528 Frankfurt/Main,
>Germany
>Email: <mailto:[log in to unmask]>[log in to unmask]