Nick,
I have used both Alexa 488 and Alexa 568 conjugated secondary antibodies
(I conjugate them myself onto antibodies from Jackson Immunoresearch).
Under the conditions I use, Alexa 488 is approximately as bright as FITC
or Oregon Green 488 to begin with and is much more resistant to
fading/bleaching than FITC. I often do confocal microscopy with my
samples, so they are getting illuminated with a lot of light, but I do
not see much bleaching. Also, if I seal my samples, they remain bright
for many days. We routinely use the Alexa 488 as our choice for green
in the lab now. The Alexa 568 does not seem to be quite as bright as
Cy3; both dyes are very bleaching/fading resistant in my prep. I like
to use the Cy3 if I am doing fluorescent microscopy with a regular
fluorescent scope. I use the Alexa 568 if I am going to use the confocal
for my imaging (since the confocal I use has a 568 line, the Alexa 568
is very slightly brighter than Cy3 with the laser).
Are you using an antifade reagent in your mounting medium? If you are
having a lot of trouble with fading, you might try changing your
reagent. I know that different reagents perform differently with
different dyes, mounting media, and preparations. I use n-propyl gallate
with the Alexa dyes in a PBS/glycerol mounting media with a lot of
success. Lance Ladic has a great web page with a discussion of
different antifade reagents:
http://www.cs.ubc.ca/spider/ladic/overview.html
Good luck,
Janet
Nick Owen wrote:
>> Hi all,
>> I have just started using Molecular Probes Alexa (www.probes.com)
> series of flurescent dyes for immunolocalisation and am finding they
> fade out very quick, in about 10-15 seconds! I was wondering if anyone
> else out there has used these dyes yet?
>> Cheers
>> Nick
>nick_owen at wols.demon.co.uk
