>Confocalists,
>What is the best way to stain murine cells with monoclonal antibodies and
>eliminate nonspecific binding of the anti-mouse fluorochrome conjugated
>secondary antibody?
>Thanks in advance.
>
>Steven Woodard
>Confocal Microscopy Facility
>Georgia Institute of Technology

Hey Folks --

        Probably the most straight-forward way is to directly conjugate the
monoclonal to a fluorophore.  The Amersham "fluorolink" kits are handy, and
can be used to link the bright cyanine dyes (Cy2, Cy3, or Cy5) onto the
antibody.  Molecular Probes also sells reactive forms of many fluorophores.

        The disadvantage to this technique is that you may not get as much
signal as you are used to seeing with secondary antibodies, due to a lack
of amplification.  A potential way around this is to use an
anti-fluorophore antibody (Mol. Probes has a very nice rabbit anti-FITC).
Thus the fluorophore on the monoclonal acts as a hapten that can be
visualized using standard indirect techniques.  Biotin is another hapten
that can be conjugated to the monoclonal and detected using either
anti-biotin or fluorescent avidin.

The usual disclaimer:   I have no commertial interest in the products
mentioned above, I just found them useful.

Happy Labeling,  Greg.

Greg Martin
Light/Confocal Microscopy Specialist
Biological Imaging Service
The Jackson Laboratory
TJL Box 43
600 Main Street
Bar Harbor. ME  04609

207-288-6191