fluorescein versus digoxigenin labled antisense riboprobes

Leon Parker mrclean at itsa.ucsf.edu
Tue Dec 17 12:32:34 EST 1996


Hello all--

I have generated both fluorescein-labeled and digoxigenin-labeled
antisense riboprobes for use in the two-color, whole-mount in situ
hybridization analysis of zebrafish embryos.  In control experiments, I
have performed side by side comparisons between antisense probes
transcribed from the same template (a cDNA for a gene that is highly
expressed in the embryonic stages we are examining) and labeled with
either DIG or fluorescein.  I have repeatedly observed that the
digoxigenin-labeled probes yield consistently better results than the
fluorescein-labeled probes.  I have made this observation despite making
sure that probe concentration and quality were equally matched.  I have
also have also attempted to detect the fluorescein-labeled probes using
anti-fluorescein Fab-alkaline phosphatase conjugated antibodies at
titers ranging from 1:100 to :1:2000.  All of reagents (in vitro
transcription reagents, antisera, etc.) are from Boehringer Mannheim.  
Has anyone made similar observations?  Any ideas as to why we see a
difference?  Could biotinylated ribonucleotides be used in conjunction
with streptavidin-alkaline phosphatase to do these experiments?  

Any information/advice/insights you can provide would be greatly
appreciated.

Sincerely, 

--Leon Parker

------- Leon Parker
        Postdoctoral Fellow
        (Laboratory of Didier Y. R. Stainier)
        University of California, San Francisco
        Department of Biochemistry & Biophysics
        Room S-960 Box 0554
        San Francisco, CA  94143-0554
        Lab phone:  (415) 502-5680
        FAX:  (415) 476-3892
        e-mail:  mrclean at itsa.ucsf.edu




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