In reply to Richard Edelman,

We have found that the Alexa dyes (when conjugated to a number of different antibodies) give higher levels of fluorescence and low amounts of fading when mounted in a PBS mount (Phosphate Buffered Saline). Simple glycerol, or a number of antifade reagents made matters worse! We have not tried the "Prolong" antifade mentioned by Jill Hendrickson from Molecular Probes - their data shows that Prolong should work well with Alexa dyes.

Propidium iodide also gives excellent fluorescence using a simple PBS mount, although it does show more fading than the Alexa dyes under the same conditions.

Good luck, Alan Hibbs

BIOCON, specialists in confocal microscopy
7 Walhalla Drive, Ringwood East VIC 3135 Australia      phone: 61 3 9876 9822
Dr. Alan R. Hibbs


-----Original Message-----
From:   Richard E. Edelmann [SMTP:[log in to unmask]]
Sent:   Saturday, May 27, 2000 3:31 AM
To:     [log in to unmask]
Subject:        Alexa 488 fading too fast

        We seem to be having a problem with Alexa 488 fading out *Really* fast (i.e.
under 20 seconds).

(1) Alexa 488 Goat Anti-rabbit (primary anti-body against a nuclear protein in
Arabidopsis). Preparation at pH 8.0.  DABCO antifade.  {Counter staining with
Propidium Iodide and it is also fading fast.  Bad DABCO?}

(2) In comparison have done experiments with Alexa 488 Goat anti-Mouse (mRNA
labeling), again DABCO antifade.  The Alexa signal was stronger and signal lasted
much longer (minutes).

        Any suggestions?

        Thanks


Richard E. Edelmann, Ph.D.
Electron Microscopy Facility Supervisor
352 Pearson Hall
Miami University, Oxford, OH 45056
Ph: 513.529.5712        Fax: 513.529.4243
E-mail: [log in to unmask]

"RAM disk is NOT an installation procedure."



BIOCON, specialists in confocal microscopy
7 Walhalla Drive, Ringwood East VIC 3135 Australia      phone: 61 3 9876 9822
Dr. Alan R. Hibbs